Type I interferons (IFNs), pleiotropic cytokines with antiviral, antiproliferative, apoptotic, and immunoregulatory functions, are efficacious in the treatment of malignancies, viral infections, and autoimmune diseases. Binding of these cytokines to their cognate receptor leads to activation of the Jak-signal transducers and activators of transcription (STAT) signaling pathway and altered gene expression. This signal pathway has been intensely studied using human IFN-α2 and IFN-β. However, there are over 14 human IFN-α subtypes and over 10 murine IFN-α subtypes, with a single IFN-β subtype in both species. J2E cells are immortalized at the proerythroblast stage of development and produce a rapid and fatal erythroleukemia in vivo. These cells retain the ability to respond to erythropoietin in vitro by proliferating, differentiating, and remaining viable in the absence of serum. Here, we show that J2E cells are also functionally regulated differentially by IFN subtype treatment in vitro. A novel finding was the selective activation of STAT and mitogen-activated protein kinase (MAPK) molecules by different subtypes binding the IFN receptor. These findings indicate distinct effects for individual type I IFN subtypes, which are able to differentially activate members of the STAT and MAPK family. Finally, we investigated the efficacy of IFN naked DNA therapy in treating J2E-induced erythroleukemia in athymic nude mice. IFN subtypes differentially regulated the onset of erythroleukemia with delayed onset and increased survival, possibly via a reduction in cell viability, and enhanced antiproliferative and apoptotic effects observed for IFNA6 and IFNA9treatment, respectively. Moreover, these data highlight the necessity to choose the best IFN subtype in disease treatment.

Erythroleukemias represent the highly malignant M6 subtype of acute myeloid leukemia.1 The J2E cell line, immortalized at the proerythroblast/basophilic erythroblast stage of erythroid development,2 induces a rapid and fatal erythroleukemia in mice.3 However, while these cells are immortalized, they are still capable of normal signaling in response to erythropoietin (Epo) stimulation. They maintain all biologic responses to Epo by differentiating biochemically with hemoglobin production and undergoing cellular alterations with a percentage of cells enucleating to form mature reticulocytes.2,4-7 J2E cells also display increased proliferation and enhanced viability in the absence of serum as a result of Epo stimulation.2,4 5 

Type I interferon (IFN) subtypes are clinically effective in the treatment of disease conditions such as hepatitis, hairy cell leukemia, condyloma acuminatum, multiple sclerosis, and Kaposi sarcoma.8-11 These IFNs have also proven effective in the treatment of both myelogenous and metastatic tumors,12,13and IFN-α has the ability to normalize hemoglobin levels in anemic patients.14 Subtype diversity for treatment is limited however to human IFN-α2 (huIFN-α2)15 and huIFN-β (IFN-β-1b; IFN-β-1a).

The type I IFNs have been attributed to multiple and diverse functions in the immune response. This multigene family has over 14 IFN-α subtypes in humans and 10 IFN-α subtypes in mice, with a single IFN-β subtype for each. The murine and human IFN gene families are highly analogous.16 Induced early in the immune response, IFNs stimulate antiviral,17,18antiproliferative,19 and apoptotic activity,20 as well as have numerous immunomodulatory effects. Type I IFNs regulate dendritic cell major histocompatibility complex (MHC) expression and maturation,21-24activate natural killer (NK) cells,25 induce bystander T-cell proliferation,26 selectively induce clonal CD8+ T-cell expansion,26 and skew the immune response to a Th1-like response.27 28 

At the cellular level, type I IFN binds the IFN-α receptor 2 (IFNAR2) subunit29 instigating its association with the IFNAR1 subunit.30 The preassociated Jak kinases, Jak1 and Tyk2, are subsequently cross-phosphorylated.31,32Phosphorylation of signal transducers and activators of transcription 2 (STAT2) occurs, which forms a heterodimer with STAT1 and phosphorylation of the latter. Phosphorylation of other STATs in response to IFN-α include STAT3,33 STAT4,34STAT5, and STAT6.35,36 Phosphorylation of STAT4 however is not generally reported in the murine system and is believed to be due to a microsatellite insertion in Stat2.37 Type I IFN induces the formation of both homodimers (STAT1,38STAT3,39 and STAT5) and heterodimers (STAT1/STAT238 and STAT1/STAT340). These STAT dimers are translocated to the nucleus where they bind type I IFN-stimulated response elements leading to gene transcription. Downstream STAT1/STAT2 heterodimers bind p48 to form the mature IFN-stimulated gene factor 3 (ISGF3) complex, a p38 mitogen-activated protein kinase (MAPK)–dependent process.41,42 During signaling for growth inhibition, IFN-α activates the Rac1/p38 MAPK pathway,43 while recombinant huIFN-α2 has been shown to activate JNK-1 and p38 MAPK in apoptotic cell death.44 Alternatively, IFN-α activation of p42 MAPK is essential for cell proliferation.45 

Previously, J2E cells stimulated with mixed murine IFN-α (muIFN-α), IFN-α1, and IFN-α4 differed in their antiproliferative capacity,46 while levels of Epo-induced differentiation were maintained.47 In this report, we investigated the differential antileukemic properties of a panel of 7 type I IFN subtypes (-α1, -α2, -α4, -α5, -α6, -α9, and -β) using delivery of an expression vector encoding the IFN subtype gene. Individual IFN subtypes were studied in vitro for effect on erythroid cell proliferation, differentiation, viability, and apoptosis. Members of the STAT and MAPK signaling families in J2E cells were examined for differential activation/phosphorylation in response to individual IFN subtypes. Finally, the efficacy of IFN naked DNA treatment was assessed in the development of J2E cell-induced erythroleukemia in vivo.

Cell culture

J2E cells2 were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (GIBCO BRL, Mount Waverly, Victoria, Australia) at 37°C in a humidified atmosphere of 5% CO2. Cells were stimulated with IFN (1-1000 IU/mL) determined by antiviral bioassay, as indicated. For signaling and viability experiments, cells were washed 3 times to be free of fetal calf serum (FCS) and incubated 5 hours at 37°C with 5% CO2 prior to stimulation with interferon. Cell viability was determined by 0.4% eosin exclusion, while hemoglobin production was detected by benzidine staining.48 

Measurement of apoptosis

Apoptosis was measured by TdT-mediated dUTP nick end labeling (TUNEL) assay (Apoptosis Detection Kit; TA200; R&D Systems, Minneapolis, MN) and by DNA integrity.49TUNEL assays were performed according to the manufacturer's instructions, staining with anti-BrdU antibody followed by streptavidin horseradish peroxidase and diaminobenzidine (DAB) substrate development. Cells were counterstained with hematoxylin, mounted, and viewed with light microscopy. DNA integrity was determined by extraction of DNA by the proteinase K method.50 DNA concentration was determined by spectrophotometry at 260 nm before electrophoresis using a 1% agarose gel.

Protein analyses

Proteins were extracted in lysis buffer (150 mM NaCl2, 20 mM Tris, pH 7.5, 1% Triton X-100 [vol/vol], 40 mM Na4P2O7, 1 mM Na3VO4, 50 mM NaF, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM benzamidine) and samples (100 μg) immunoblotted with antiphospho-STAT1, -STAT3, -STAT5, and -STAT6 antibodies (phospho-STAT antibody sampler, no. 9914; New England Biolabs, Beverly, MA) or antiphospho-JNK, -p38, -p42, or -p44 MAPK antibodies (phospho-MAPK antibody sampler, no. 9910; New England Biolabs). Proteins (2 mg/mL) were immunoprecipitated with anti-STAT2 and -STAT4 antibodies (sc-476, sc-478; Santa Cruz Biotechnology, Santa Cruz, CA) and immunoblotted with antiphosphotyrosine IgG (no. 05-321, clone 4G10; Upstate Biotechnology, Lake Placid, NY). Antibody binding was determined by enhanced chemiluminescence (ECL; Pharmacia Biotech, Buckinghamshire, United Kingdom) and visualized by autoradiography. Levels of p42 MAPK (sc-154; Santa Cruz Biotechnology) were used as a loading control in each experiment.

Northern analysis of double-stranded (ds)RNA-activated protein kinase (PKR) and 2′5′ oligoadenylate synthetase (2′5′OAS)

Total RNA from J2E cells was extracted using TRIZOL (GIBCO BRL), in accordance with the manufacturer's instructions, and RNA samples (20 μg) were electrophoresed on formaldehyde-agarose gels prior to transfer to nylon membranes (MSI, Westborough, MA) in 10 × SSC (1.5 M sodium chloride, 150 nM trisodium citrate, pH 7.0). Full-length murine cDNA (100 ng) for PKR (1.4 kilobase [kb]) and 2′5′OAS (1.8 kb), obtained from plasmids provided by B. Williams (Cleveland Clinic Foundation, Cleveland, OH), or glyceraldehyde phosphate dehydrogenase (GAPDH) was 32P-labeled using a Gigaprime labeling kit (Geneworks, Adelaide, Australia). Membranes were hybridized to α-32P-labeled probes at 65°C in 6 × sodium chloride tri-sodium (SSC); 10 nM sodium phosphate buffer, pH 7.0; 1 mM ethylenediaminetetraacetic acid; 1 × Denhardt; 5% dextran sulfate (wt/vol); 0.1% sodium dodecyl sulfate (SDS); and 100 μg/mL herring sperm DNA for 18 hours. Following hybridization, membranes were washed to 0.2 × SSC/0.1% SDS (wt/vol) and visualized by autoradiography.

Preparation of interferons

IFN DNA constructs comprising IFN-A1, -A2, -A4, -A5, -A6, -A9, and -B genes cloned into the mammalian expression vector pkCMVint.polylinker (VICAL, San Diego, CA) have been described elsewhere.51 Plasmid DNA was prepared from Terrific Broth culture of transformed Escherichia coli (DH5α; Life Technologies, Grand Island, NY) using standard extraction procedures with LiCl precipitation. Concentration and purity of plasmid DNA was determined by spectrophotometric analysis at 260 nm, with a 260:280 nm ratio of 1.6 or higher.

IFN protein was prepared from transfected COS-7 cells expressing the individual IFN DNA constructs. In brief, COS-7 cells were calcium phosphate-transfected with 20 μg plasmid DNA.52 Cells were cultured in 5% FCS or serum-free conditions for 24 or 48 hours. Supernatants were harvested, pH 2 acid–treated to remove acid-labile IFNs, and IFN content determined by titration against the international standard IFN-α/β (Lee Biomolecular, San Diego, CA; 1000 IU/mL) using encephalomyocarditis virus–infected L929 cells in a bioassay as previously described.53 All COS-7 cells transfected with the IFN-A/B transgenes showed secretion of various amounts of IFN-α (pg/mL) by enzyme-linked immunosorbent assay (ELISA) using an antimurine IFN-α antibody kit (PBL Biomedical Laboratories, Piscataway, NJ). Though cells transfected with the blank vector or IFN-βtransgene secreted nominal amounts of IFN-α. The 6 alpha IFN subtypes investigated in this study were equated to pg/1000 IU based on these assays (IFN-α1, 2583 pg; IFN-α2, 1549 pg; IFN-α4, 1294 pg; IFN-α5, 3111 pg; IFN-α6, 371pg; and IFN-α9, 94 pg).

Naked DNA treatment

Specific pathogen-free, 4-week-old homozygousnu/nu BALB/c mice (Animal Resources Centre, Murdoch, Western Australia) were inoculated bilaterally in each tibialis anterior (TA) muscle with 100 μg plasmid DNA (vehicle or encoding type I IFN subtype) in a 25 μL volume, as previously described.51,53 Circulating expression levels of IFN transgenes in vivo have previously been found to be similar, with an average of 20 IU/mL sera.49 

Erythroleukemia

At 14 days following DNA treatment, mice were inoculated with 1 × 106 J2E cells by the intravenous route as described previously,3 and culled as they became moribund. Hematocrit readings were taken via tail-vein bleeds prior to culling, and spleen and liver weights were determined at autopsy.

Statistical analyses

Significant differences between means were determined using a 2-tailed Student t test (P ≤ .05). Statistical significance between cumulative survival rates of animals was determined by the Cox proportional hazards regression model.

Antiproliferative effects of IFN stimulation of J2E cells

Type I IFNs were initially examined for their antiproliferative effect on J2E cell growth via IFN dose-response analysis (1-1000 IU/mL) during 48 hours of cell culture. All IFN subtypes examined (IFN-α1, -α2, -α4, -α5, -α6, -α9, and -β) reduced J2E cell number (Figure 1) with IFN-α1, -α4, -α5, and -α6 acting in a dose-dependent manner. Interestingly, suppression of J2E cell growth required a higher dose of IFN-α2 and -β (100-1000 IU/mL) while very low concentrations of IFN-α9 (1 IU/mL) significantly reduced cell number after 48 hours of culture.

Fig. 1.

Type I IFN dose-response analysis of J2E cell growth.

J2E cells were seeded at 5 × 104 cells/mL and stimulated with IFN-α1, -α2, -α4, -α5, -α6, -α9, or -β (1-1000 IU/mL) as indicated. Cell number was determined by eosin exclusion at 48 hours of culture (statistical significance compared with unstimulated J2E cells, *P ≤ .05; **P ≤ .01; mean ± SE, n = 3).

Fig. 1.

Type I IFN dose-response analysis of J2E cell growth.

J2E cells were seeded at 5 × 104 cells/mL and stimulated with IFN-α1, -α2, -α4, -α5, -α6, -α9, or -β (1-1000 IU/mL) as indicated. Cell number was determined by eosin exclusion at 48 hours of culture (statistical significance compared with unstimulated J2E cells, *P ≤ .05; **P ≤ .01; mean ± SE, n = 3).

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Individual IFN subtype efficacies of J2E cell differentiation

J2E cells respond to Epo stimulation by synthesizing hemoglobin. Therefore we next determined the effect of IFN stimulation, in the absence of Epo, on differentiation of J2E cells (Figure2). IFN-α4 was the only IFN subtype that significantly enhanced J2E cell differentiation at 100 IU/mL, while stimulation at 1000 IU/mL induced significant increases in differentiation of J2E cells with all the IFNs subtypes (up to 5.5-fold with IFN-α1) except IFN-α9 and -β. The maturation of J2E cells in the presence of IFN was therefore dependent on the IFN subtype chosen.

Fig. 2.

Effect of type I IFNs on J2E erythroid cell differentiation.

J2E cells were seeded at 5 × 104 cells/mL and stimulated with IFN subtypes at (A) 100 and (B) 1000 IU/mL. Hemoglobin production was measured by benzidine staining at 48 hours of culture. (statistical significance compared with unstimulated J2E cells, *P ≤ .05; **P ≤ .01; mean ± SE, n = 3).

Fig. 2.

Effect of type I IFNs on J2E erythroid cell differentiation.

J2E cells were seeded at 5 × 104 cells/mL and stimulated with IFN subtypes at (A) 100 and (B) 1000 IU/mL. Hemoglobin production was measured by benzidine staining at 48 hours of culture. (statistical significance compared with unstimulated J2E cells, *P ≤ .05; **P ≤ .01; mean ± SE, n = 3).

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Differential IFN subtype efficacies of J2E cell viability and apoptosis

Since type I IFNs are known inducers of apoptotic cell death, we therefore investigated the effect of the IFN subtypes on J2E cell viability and apoptosis. Cells stimulated with 100 IU/mL of IFN-α1, -α4, -α5, and -α6 all showed a significant reduction in J2E cell viability by 75.6%, 55.3%, 61.8%, and 63.8%, respectively (Figure 3). In direct contrast, however, when using higher concentrations of IFN (1000 IU/mL), only IFN-α6 and -β significantly reduced J2E cell viability. We speculate that the reduced level of cell death at high IFN concentration may be due to the necessity for maintaining circulating erythrocyte numbers during distress or infection.

Fig. 3.

Effect of type I IFNs on J2E erythroid cell viability.

J2E cells were washed free of serum, seeded at 3 × 105cells/mL, and stimulated with IFN subtypes at (A) 100 or (B) 1000 IU/mL. Percentage viable cells was determined by eosin exclusion at 48 hours of culture (statistical significance compared wtih unstimulated J2E cells, *P ≤ .05; **P ≤ .01; mean ± SE, n = 3).

Fig. 3.

Effect of type I IFNs on J2E erythroid cell viability.

J2E cells were washed free of serum, seeded at 3 × 105cells/mL, and stimulated with IFN subtypes at (A) 100 or (B) 1000 IU/mL. Percentage viable cells was determined by eosin exclusion at 48 hours of culture (statistical significance compared wtih unstimulated J2E cells, *P ≤ .05; **P ≤ .01; mean ± SE, n = 3).

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Next, IFN-induced apoptosis was examined in J2E cells by TUNEL assay following stimulation with 100 IU/mL IFN (Figure 4A-B). A significant enhancement in the rate of apoptosis was observed for IFN-α9 (71.3%) with a moderate increase detected for IFN-β (57.6%) in contrast to control, unstimulated cultures (44.7%). Notably IFN-α5 significantly reduced apoptotic cell death to 34.3%, a 10.4% reduction compared with control cells. These results indicate differential apoptotic potential of individual IFN subtypes in J2E cells.

Apoptosis was also measured by DNA degradation in response to J2E cell stimulation with 1000 IU/mL of the IFN subtypes (Figure 4C). Interestingly, DNA fragmentation at 6 hours after IFN stimulation was minimal for all subtypes examined (IFN-α5 at 6 hours, not done), with the exception of IFN-α9. At 24 hours, stimulation with IFN-α2 and -α4 (1000 IU/mL) increased while IFN-β reduced DNA fragmentation. These data indicate that while lower doses of IFN-α9 or IFN-α5 may enhance or reduce apoptosis, it appears that high concentrations of IFN potentiate the onset of apoptosis of J2E cells cultured in the absence of serum.

Fig. 4.

Effect of type I IFNs subtypes on J2E cell apoptosis.

(A) Apoptosis of J2E cells in response to stimulation with IFN subtypes (100 IU/mL), in the absence of serum, at 24 hours by TUNEL assay. Apoptotic cell bodies stained with diaminobenzidine (DAB, black) and cells counterstained with hematoxylin (gray). Original magnification, × 40. (B) The percentage of positive staining apoptotic cells was determined by light microscopy. (Statistical significance compared with unstimulated J2E cells, *P ≤ .05, mean ± SE, n = 3.) (C) At 6 and 24 hours after stimulation with 1000 IU/mL IFN, in the absence of serum, DNA fragmentation (≤ 6 kb) was determined in J2E cells. Control cultures were unstimulated.

Fig. 4.

Effect of type I IFNs subtypes on J2E cell apoptosis.

(A) Apoptosis of J2E cells in response to stimulation with IFN subtypes (100 IU/mL), in the absence of serum, at 24 hours by TUNEL assay. Apoptotic cell bodies stained with diaminobenzidine (DAB, black) and cells counterstained with hematoxylin (gray). Original magnification, × 40. (B) The percentage of positive staining apoptotic cells was determined by light microscopy. (Statistical significance compared with unstimulated J2E cells, *P ≤ .05, mean ± SE, n = 3.) (C) At 6 and 24 hours after stimulation with 1000 IU/mL IFN, in the absence of serum, DNA fragmentation (≤ 6 kb) was determined in J2E cells. Control cultures were unstimulated.

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IFN subtypes activate different members of the STAT family

Having established different IFN effects on J2E cell proliferation, differentiation, viability, and apoptosis, we next examined signaling pathways that may be involved in this process. First, we analyzed the activation of STAT molecules in response to stimulation with the IFN subtypes. Phosphorylation of STAT molecules in response to IFN (100 IU/mL) was examined using antiphospho-STAT antibodies. Tyrosine phosphorylation of STAT1 was induced in response to IFN-α1, -α2, -α4, and -α5, while tyrosine phosphorylation of STAT3 was induced in response to IFN-α1, with partial activation in response to IFN-α4, -α5, and -β at 15 minutes after stimulation (Figure 5A). Tyrosine phosphorylation of STAT5a, STAT5b, or STAT6 (data not shown) and serine phosphorylation of STAT3 was not detected at this low concentration of IFN (Figure 5A).

Fig. 5.

Phosphorylation of STAT and MAPK family members in response to IFN stimulation of J2E cells.

Cells were serum starved for 5 hours prior to stimulation with IFN subtypes (100-1000 IU/mL). Total cell protein (100 μg) was extracted at the indicated times and immunoblotted with (A) antiphospho-STAT1 (STAT1-Y) and antiphospho-STAT3 (STAT3-Y, STAT3-S) antibodies; (B) antiphospho-STAT1 (STAT1-Y), antiphospho-STAT3 (STAT3-Y, STAT3-S), antiphospho-STAT5 (STAT5a-Y), and antiphospho-STAT6 (STAT5b-Y) antibodies; and (C) antiphospho-p38, antiphospho-p42, and antiphospho-p44 MAPK antibodies. Immunoblot with anti-p42 MAPK antibodies indicates protein loading.Y- denotes tyrosine; S-, serine.

Fig. 5.

Phosphorylation of STAT and MAPK family members in response to IFN stimulation of J2E cells.

Cells were serum starved for 5 hours prior to stimulation with IFN subtypes (100-1000 IU/mL). Total cell protein (100 μg) was extracted at the indicated times and immunoblotted with (A) antiphospho-STAT1 (STAT1-Y) and antiphospho-STAT3 (STAT3-Y, STAT3-S) antibodies; (B) antiphospho-STAT1 (STAT1-Y), antiphospho-STAT3 (STAT3-Y, STAT3-S), antiphospho-STAT5 (STAT5a-Y), and antiphospho-STAT6 (STAT5b-Y) antibodies; and (C) antiphospho-p38, antiphospho-p42, and antiphospho-p44 MAPK antibodies. Immunoblot with anti-p42 MAPK antibodies indicates protein loading.Y- denotes tyrosine; S-, serine.

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Stimulation of J2E cells with IFN at 1000 IU/mL was next examined for phosphorylation of STAT molecules (Figure 5B). Tyrosine phosphorylation of STAT1 was induced in J2E cells in response to all 7 IFN subtypes. IFN-α1 and -α4 (1000 IU/mL) induced tyrosine phosphorylation of STAT5a and STAT5b. Tyrosine phosphorylation of STAT3 was not detected with IFN stimulation at 1000 IU/mL, however, serine phosphorylation of STAT3 was detected in response to all subtypes with an elevated level of phosphorylation for IFN-α9 and IFN-β (Figure 5B). STAT2 was tyrosine phosphorylated in response to all subtypes at both IFN concentrations (100 and 1000 IU/mL), while phosphorylation of STAT4 was not detected (data not shown).

In summary, stimulation of J2E cells with the IFN subtypes led to differential phosphorylation/activation of STAT molecules that altered with the concentration of stimulating cytokine. While STAT1 and STAT2 were phosphorylated at high and low concentrations for all IFN subtypes examined, STAT3 tyrosine and serine phosphorylation was subtype- and concentration-dependent. Tyrosine phosphorylation has been found to be important for dimerization of STAT proteins, whereas serine phosphorylation is required for maximum transcriptional activation mediated by some STAT proteins, including STAT3.54 The specific activation of STAT5a and STAT5b with IFN subtypes at 1000 IU/mL was an unexpected finding and interestingly correlated with increased hemoglobin-positive staining in J2E cells stimulated with IFN. Indeed, STAT5b activation has been shown to be critical for differentiation of erythroid cells.55 56 

Differential activation of MAPK family members by IFN-α and -β subtypes

As stimulation of J2E cells with low concentrations of IFN (100 IU/mL) activated a limited panel of STAT molecules, we therefore examined the phosphorylation of MAPK family members in response to IFN stimulation. Phosphorylation of JNK was detected upon stimulation with IFN-β only and not with any of the other IFN-α subtypes (data not shown). However, p38 MAPK was activated with all of the IFN-α subtypes but not with IFN-β, while p42 and p44 MAPK were activated by all IFN-α and IFN-β subtypes (Figure 5C). Thus the MAPK family members displayed distinct activation patterns compared with the STAT molecules.

Type I IFNs activate ISGs

Type I IFNs are known to activate interferon-stimulated genes (ISGs), therefore we examined the expression of PKR and 2′5′OAS. Stimulation of J2E cells with all IFN subtypes led to the activation of PKR by 5 hours, with a higher expression of PKR detected at 2 hours for IFN-α9 and IFN-β subtypes (Figure6A). Similarly, all IFN subtypes activated 2′5′OAS over 2 to 5 hours (Figure 6B). These data indicate that IFN (100 IU/mL) effectively induced gene expression of PKR and 2′5′OAS in the J2E cell system.

Fig. 6.

Type I IFNs stimulate expression of antiviral ISGs in J2E cells.

J2E cells were seeded at 1 × 105 cells/mL, stimulated with type I IFNs (100 IU/mL), and RNA extracted at indicated time points. RNA (20 μg) was electrophoresed on formaldehyde gels, Northern blotted, and hybridized with (A) PKR or (B) 2′5′OAS. GAPDH loading control as indicated.

Fig. 6.

Type I IFNs stimulate expression of antiviral ISGs in J2E cells.

J2E cells were seeded at 1 × 105 cells/mL, stimulated with type I IFNs (100 IU/mL), and RNA extracted at indicated time points. RNA (20 μg) was electrophoresed on formaldehyde gels, Northern blotted, and hybridized with (A) PKR or (B) 2′5′OAS. GAPDH loading control as indicated.

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IFN therapy for J2E cell-induced erythroleukemia

J2E cells induce a rapid fatal erythroleukemia, following intravenous injection into mice, which is characterized by severe anemia and hepatosplenomegaly.3 Previously, it has been shown that altering signaling molecules can impede the progression of erythroleukemia induced by J2E cells.6,57 Therefore, since the IFN subtypes varied in their antigrowth, differentiation, cell viability, apoptotic response, and induced altered signaling pathways in J2E cells, we investigated their therapeutic potential for J2E cell-induced erythroleukemia. Mice were injected with IFN plasmid DNA into the TA muscle 2 weeks prior to intravenous injection of J2E cells, thus allowing expression of the transgene in vivo.51Animals were culled as they became moribund and the degrees of anemia, hepatosplenomegaly, and tumor burdens were monitored.

All IFN-α subtypes delayed the onset of mortality compared with the death rate of mice inoculated with the control vector (vehicle). Notably, IFN-A1, IFN-A6, and IFN-A9 delayed the onset of mortality by 12 days compared with the vehicle-treated mice, which commenced dying at day 35 (P ≤ .1, Figure7). In addition, survival rates of mice following DNA treatment with IFN-A6 and IFN-A9were 60% (P ≤ .1) and 40% (P ≤ .1), respectively, compared with 0% survival of control mice. In contrast,IFN-A2 increased the rate of death induced by J2E cells with all mice killed by day 49. IFN-B treatment, however, neither delayed onset of mortality nor increased survival rate compared with treatment with the vehicle. Thus, specific IFN-α subtypes were effective in extending the time to disease onset and life span of leukemia-cell recipients.

Fig. 7.

Effect of IFN transgene expression on the onset of J2E cell-induced erythroleukemia.

Mice were vaccinated bilaterally with 100 μg IFN DNA in the TA muscle and 14 days later injected with 1 × 106 J2E cells via the tail vein. The cumulative survival was calculated as the mice became moribund (n = 5-7). Vehicle-treated mice were injected with vector DNA without IFN gene cassette.

Fig. 7.

Effect of IFN transgene expression on the onset of J2E cell-induced erythroleukemia.

Mice were vaccinated bilaterally with 100 μg IFN DNA in the TA muscle and 14 days later injected with 1 × 106 J2E cells via the tail vein. The cumulative survival was calculated as the mice became moribund (n = 5-7). Vehicle-treated mice were injected with vector DNA without IFN gene cassette.

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Reduced hematocrit readings indicating anemia and the presence of hepatosplenomegaly are indicators of erythroleukemia development. Therefore, hematocrit readings, and liver and spleen weights for normal, control, and IFN-treated mice were monitored (Table1). The vehicle-treated group developed typical J2E-induced erythroleukemia with characteristic hepatosplenomegaly. In the moribund mice, hepatomegaly was apparent forIFN-A1 (P ≤ .05), IFN-A4(P ≤ .05), and IFN-B (P ≤ .01) DNA treatment while splenomegaly was observed for IFN-A1(P ≤ .01), IFN-A2 (P ≤ .05),IFN-A6 (P ≤ .05), IFN-A9(P ≤ .05), and IFN-B (P ≤ .01) DNA treatment. However, healthy mice sampled at day 83 (end of experiment) had normal spleen weights for IFN-A6 andIFN-A9 DNA treatment groups, 0.16 g ± 0.05 g and 0.34 g ± 0.07 g, respectively. As expected, hematocrit readings were reduced at time of death for vehicle-, IFN-A2, IFN-A4, IFN-A5, and IFN-B DNA-treated mice compared with normal mice. Interestingly, hematocrit readings for DNA treatment with effective subtypes IFN-A1, IFN-A6, andIFN-A9 were not significantly altered from control animals. Indeed, hematocrit readings for survivors of IFN-A6 andIFN-A9 DNA treatment groups at day 83 were normal, at 56.6% ± 1.3 and 48.9% ± 1.1, respectively.

Table 1.

Liver, spleen, and hematocrit readings near death in J2Eand IFN-treated mice

Treatment groupLiver, gSpleen, gHematocrit, %
Control 1.30 ± 0.02 0.135 ± 0.007 52.5 ± 0.8 
Vehicle 1.90 ± 0.12* 0.636 ± 0.155* 39.5 ± 2.6 
A1 1.79 ± 0.04* 0.543 ± 0.072 44.9 ± 2.9 
A2 1.60 ± 0.24 0.993 ± 0.222* 36.7 ± 2.1 
A4 2.27 ± 0.14* 0.666 ± 0.229 44.1 ± 1.5* 
A5 2.59 ± 0.58 1.130 ± 0.336 36.1 ± 2.7* 
A6 1.99 ± 0.25 0.902 ± 0.232* 42.0 ± 5.8 
A9 1.58 ± 0.10 0.616 ± 0.232* 42.8 ± 2.8 
1.83 ± 0.09 0.392 ± 0.066 44.1 ± 2.5* 
Treatment groupLiver, gSpleen, gHematocrit, %
Control 1.30 ± 0.02 0.135 ± 0.007 52.5 ± 0.8 
Vehicle 1.90 ± 0.12* 0.636 ± 0.155* 39.5 ± 2.6 
A1 1.79 ± 0.04* 0.543 ± 0.072 44.9 ± 2.9 
A2 1.60 ± 0.24 0.993 ± 0.222* 36.7 ± 2.1 
A4 2.27 ± 0.14* 0.666 ± 0.229 44.1 ± 1.5* 
A5 2.59 ± 0.58 1.130 ± 0.336 36.1 ± 2.7* 
A6 1.99 ± 0.25 0.902 ± 0.232* 42.0 ± 5.8 
A9 1.58 ± 0.10 0.616 ± 0.232* 42.8 ± 2.8 
1.83 ± 0.09 0.392 ± 0.066 44.1 ± 2.5* 

As mice became moribund, blood was harvested by tail bleed; spleen and liver were removed immediately following death and weighed. Control mice received no treatment and were killed after the last mouse became moribund.

Values represent mean ± standard error.

*

Significance compared with normal mice, P < .05

P ≤.01

Numerous tumors were observed in the lymph nodes ofvehicle-, IFN-A1–, IFN-A2–, and IFN-B–treated mice (Table 2). IFN-A9treament was not associated with tumor involvement of lymph nodes. Furthermore, neither IFN-A6- nor IFN-A9DNA-treated mice showed evidence of multiple tumors that was observed in the vehicle-treated mice. Interestingly, introduction ofIFN-B also induced cachexia in 2 mice. These data therefore indicate a possible influence of IFN subtypes on tumor homing in the mice.

Table 2.

Tumor localization in J2E- and IFN-treated moribund mice

Treatment groupCachexia, nAxillary, nInguinal, nCervical, nTotal, n/N
Normal 0/5 
Vehicle (1) (1) 1/5 
A1 (3) (3) 3/6  
A2 3/4 
A4 1/5  
A5 1/4 
A6 1/5  
A9 0/6 
1(1) 1(1) 5/7 
Treatment groupCachexia, nAxillary, nInguinal, nCervical, nTotal, n/N
Normal 0/5 
Vehicle (1) (1) 1/5 
A1 (3) (3) 3/6  
A2 3/4 
A4 1/5  
A5 1/4 
A6 1/5  
A9 0/6 
1(1) 1(1) 5/7 

As mice became moribund they were killed and autopsied.

Numbers in parentheses indicate mice with multiple tumors.

The recent establishment, in this laboratory, of a large panel of murine IFN-α subtypes and IFN-β for DNA gene therapy51and IFN protein production51,58 has provided an avenue through which to explore the anticancer effects of this multigene cytokine family. The divergent antiviral effects of the IFN subtypes are evident against numerous viruses including murine cytomegalovirus,51,58 herpes simplex virus type 1 (HSV-1),59 and HSV-2.60 Furthermore, covaccination of IFN subtypes with viral vaccines produces differential protective effects on viral infection.51 To our knowledge this is the first investigation into the antileukemic effects of such a broad panel of IFN subtypes on the development of erythroleukemia.

The antiproliferative effects of type I IFNs have been established in numerous and diverse cancers and cell lineages.15,61Specifically in erythroid cells, constitutive expression of consensus type I IFN (IFN-con1) has been shown to revert the malignant phenotype in vitro as indicated by growth inhibition in culture.62Previous reports in erythroid cells have shown antiproliferative effects with mixed IFN-α, IFN-α1, and IFN-α4.46,47We report here, the antiproliferative effects of 7 type I IFNs on erythroid J2E cells. In this study, all IFN subtypes examined induced an antiproliferative response in J2E cells, with cell numbers reduced by 51.3% to 67.9%, and IFN stimulation at 100 IU/mL, which was further reduced by 66.1% to 89.4% using 1000 IU/mL. Significantly, IFN-α9 was able to effectively reduce cell proliferation at 1 IU/mL. Previously, J2E cell growth inhibition has been observed at 10 IU/mL when stimulated with mixed muIFN-α. Studies in other erythroid systems have indicated the effectiveness of as little as 0.01 IU/mL recombinant IFN-α2a in suppressing erythroblast numbers, while alternative IFNs, including recombinant IFN-α2b, hybrid IFN-α1-8, and IFN-con1, were not effective at this dose.63 

J2E cells respond to the hormone Epo with increased differentiation, with a proportion of cells enucleating to form mature reticulocytes.64 Prior findings have indicated that mixed muIFN-α (100-300 IU/mL) stimulation of J2E cells did not intervene with Epo-induced J2E cell differentiation.47 However, in this study, in the absence of Epo, IFN-α4 (100 IU/mL) was able to significantly increase the percentage of hemoglobin-positive J2E cells, while higher doses of IFN-α1 and -α4 (1000 IU/mL) induced 5-fold and 4-fold enhancement of differentiation, respectively. Interestingly, decreased cell proliferation has been shown to potentiate Epo-induced differentiation.4,65 In the absence of Epo, chemical induction of J2E cells with sodium butyrate severely restricts proliferation while stimulating hemoglobin production in up to 100% of cells.66 Data presented here indicate that stimulation of J2E cells with specific IFN subtypes elevates the number of resting hemoglobin-positive cells. However, decreased rate of cell growth alone was not sufficient to induce cell differentiation, indicating that other intracellular mechanisms are at hand in this process.

In addition to differentiation and proliferation, J2E cells respond to Epo with a third biologic function, namely enhanced viability.5 Recent reports have indicated that type II IFN (IFN-γ) promotes enhanced viability in peripheral blood cells and stimulates the survival of erythroid colony-forming cells.67 We therefore investigated the differential effects of type I IFN on the viability of J2E erythroid cells in vitro. Low concentrations (100 IU/mL) of IFN-α1, -α4, -α5, and -α6 reduced J2E cell viability, while stimulation with higher doses of IFN (1000 IU/mL) did not reduce J2E cell viability with the exception of IFN-α6 and -β treatment. In response to localized infection, IFN is secreted to stimulate a danger signal and subsequent initiation of an innate immune response.68 Elevated doses of IFN may be indicative of systemic infection, a situation in which decreased circulating erythrocytes would be detrimental to the individual. Indeed, therapy with high- dose IFN-α has proved effective in normalizing hemoglobin levels in anemic patients.14 

The IFNs are inducers of apoptosis in malignancies including herpesvirus-associated lymphomas, acute promyelocytic leukemia, non–small cell lung cancer, nonmelanoma skin cancer, and glioma.20 Early findings of growth inhibition in erythroid progenitor cells implicated an apoptotic mechanism via DNA fragmentation.69 At low IFN concentrations (100 IU/mL), apoptosis in the J2E cell was enhanced by IFN-α9, and partially but significantly reduced with IFN-α5 treatment. At higher concentrations of IFN (1000 IU/mL), all IFN-α subtypes induced DNA fragmentation. IFN-β however, had minimal apoptotic activity on J2E cells. Similarly, other researchers have shown decreased rates of hematopoietic cell proliferation in the presence or absence of apoptosis.70 71 

Stimulation of J2E cells with the type I IFNs led to differential effects at the cellular level, presumably via stimulation of various signaling cascades following activation of the IFN-α receptor (IFNAR). Surprisingly, the majority of signaling studies have used a limited range of type I IFN subtypes. Divergence between IFN-α and IFN-β signaling can be observed at the levels of Jak/STATs,72,73 Ras/Raf/MAPK,45,74,75IRS-1,76,77 and CrkL phosphorylation.73 Most studies in humans have concentrated on signal events stimulated by either unspecified IFN-α IFN-α mixtures,76,78recombinant IFN-α2,32,41-43,73,79IFN-α2c,78 IFN-α A/D,76 IFN-β, or IFN-β-1b.32,42,73-75,77-80 However, Hilkens et al81 have recently examined recombinant human IFN subtypes -α1, -α2, and -α21 for differential STAT1 activation and found that IFN-α1 was weaker in tyrosine phosphorylation of STAT1 than the other IFN subtypes. In the murine system, IFN signaling has been examined in response to IFN-α,37IFN-α/β mix,80 and IFN-β.9 Given that purified IFN-α mixture is predominantly IFN-α2,15 most of these studies compare only IFN-α2 and IFN-β subtypes.

IFN-α (500 IU/mL) in the human lymphoblastoid cell line Daudi activates STAT1, STAT2, STAT3, STAT5, and STAT6, and Fasler-Kan et al35 propose that cell type-specific activation of STATs could be responsible for cell type-specific responses to IFN-α.35 In the J2E erythroid system, while all IFN subtypes activated STAT2 regardless of IFN concentration, activation of other STAT family members was more specific to subtype and dosage. Low concentrations of IFN-α1, -α2, -α4, and -α5 phosphorylated STAT1, while high concentrations of all IFN subtypes induced STAT1 tyrosine phosphorylation and STAT3 serine phosphorylation. Further, low levels of IFN-α1 activated STAT3 tyrosine phosphorylation (partial activation by IFN-α4, -α5, and -β; 100 IU/mL), while high levels of IFN-α1 and -α4 activated STAT5a and STAT5b. The biologic relevance between serine and tyrosine phosphorylation of STAT3 via IFN signaling in J2E cells is undefined and warrants further investigation. Type I IFNs primarily bind the IFNAR2 subunit, which is thought to confer differential subtype recognition, followed by association with IFNAR1 that results in a high-affinity receptor complex.82-84 Data presented here comply with differential activation of the IFNAR complex with individual IFN subtypes; moreover, these receptor complexes led to the specific activation of downstream STAT molecules.

Many signaling cascades culminate in activation of members of the MAPK family. IFN-α has been shown to activate p38 MAPK43 and p42 MAPK,45 while IFN-β activates Raf-1 and p42 MAPK.74 All 6 IFN-α subtypes stimulated phosphorylation/activation of p38 MAPK, while only IFN-β activated JNK phosphorylation. Both IFN-α and -β induced p42 MAPK and p44 MAPK phosphorylation. Other studies have shown that activation of MAPK family members is necessary for STAT activation.43 Data presented here indicate phosphorylation of p42 MAPK and p44 MAPK; however relevance to STAT activation is an area for further investigation.

PKR has antiviral85 as well as antitumor activities86,87 and is firmly established as an IFN-inducible gene.88 PKR modulates cytokine signaling and transcriptional activation via NF-κB,89STAT,90 and c-Myc91 and is implicated in regulation of cell growth and apoptosis. Similarly, 2′5′OAS, another IFN-induced protein, is implicated in suppression of cell growth.92 All 7 type I IFN subtypes examined in the present study induced the expression of both PKR and 2′5′OAS, indicating a potential mechanism for the suppression of cell growth in the J2E erythroid system.

Gene therapy has been highlighted recently as an alternative form of antiviral and anticancer treatment. IFN DNA therapy has clearly demonstrated that type I IFNs differ in their antiviral capacity and modulation of the immune response.51,57-59,93 Other investigators have found that type I IFN is a clinical target with promising antileukemic potential. In cancer treatment, IFN-α electroporation gene therapy results in tumor regression.94 Splenic CTL activity against a murine colorectal adenocarcinoma cell line, MC38, was markedly enhanced with murine IFN-α2 transduction of such tumor cells.95 Mice with Friend virus–induced leukemia and treated with poly I:C, a strong inducer of type I IFNs, were shown to have higher numbers of NK cells, no evidence of erythroleukemic tumor cells, and prolonged survival.96 Furthermore, athymic nude mice treated with neutralizing antibodies to IFN-α/β have marked enhancement in growth of 6 different subcutaneous tumors.13,97 In this study, J2E cell–induced erythroleukemia responded differentially to type I IFN gene therapy, with IFN-A6 and IFN-A9most effective in prolonging animal survival (P ≤ .1) despite similar low levels of all IFN DNA transgenes expressed in vivo (10-40 IU/mL).51,58 Thus IFN subtype transgene expression in mice is biologically effective using the naked DNA delivery system. Indeed research by Piehler et al98suggested that “for maintaining transcription of IFN-responsive genes over a longer time period, low but continuous signaling through the IFN receptor is essential.”98(p40425) Since the subtypes of IFN-α have multiple properties, the antileukemic mechanisms for IFN-α6 and IFN-α9 may be pleiotropic and include both direct antitumor effects and indirect immunomodulatory effects (eg, NK cell activation and MHC class I expression). Interestingly at a low concentration in vitro, IFN-α6 was most potent at decreasing J2E cell viability, while IFN-α9 was superior at decreasing proliferation and enhancing apoptosis. Both of these IFN subtypes were shown to exert their effects accompanied by activation of PKR and 2′5′OAS in vitro, which are involved in down-regulation of expression of growth factors leading to antiproliferation and/or apoptosis. Alternatively, we have previously shown the immunomodulatory capacity of IFN-A6 andIFN-A9 against viral infection.51 It may be that effective subtypes stimulate the innate immune response and subsequent clearance of invasive erythroleukemic cells.

This is the first time that the individual type I IFN subtypes have been found to display differential antileukemic mechanisms. A summary of both the in vitro and in vivo antileukemic effects of the individual IFN subtypes used at low doses is shown in Table3. Taken together, the results indicate that IFN subtypes differentially regulate the onset of erythroleukemia, with significantly delayed onset and increased survival possibly via reduction in cell viability with IFN-A6, and enhanced antiproliferative and apoptotic effects with IFN-A9 DNA treatment. The in vivo data show that IFN-A6 andIFN-A9 treatment may inhibit the development of anemia and increase survival time. Interestingly, the results observed with IFN-α6 and IFN-α9 are similar to the effects we previously observed when the activity of the Epo receptor and its associated tyrosine kinase Lyn were altered in J2E cells.6 57 The consequence of manipulating these signaling molecules was an increased survival time in mice given J2E-induced erythroleukemia. In summary, our data indicate differential capacity of type I IFNs in immunomodulation of cellular responses such as proliferation, differentiation, viability, and apoptosis. Furthermore, the type I IFN subtypes, shown here to activate different subsets of STAT molecules, may allude to their specific recognition of the IFNAR. Finally, we demonstrate the efficacy of IFN gene therapy in the treatment of erythroleukemia, highlighting the differential capacity of IFN subtypes and the need for best subtype choice in disease treatment.

Table 3.

Summary of antileukemic properties for IFN subtypes

IFN subtypeA1A2A4A5A6A9B
Antiproliferative ++ 
Differentiation − ++ − − 
Decreased viability ++ ++ ++ ++ 
Apoptosis − − − − ++ −  
Delayed onset of
erythroleukemia 
++ ++ ++ − 
Increased survival
rate from
erythroleukemia 
− ++ ++ − 
IFN subtypeA1A2A4A5A6A9B
Antiproliferative ++ 
Differentiation − ++ − − 
Decreased viability ++ ++ ++ ++ 
Apoptosis − − − − ++ −  
Delayed onset of
erythroleukemia 
++ ++ ++ − 
Increased survival
rate from
erythroleukemia 
− ++ ++ − 

++ indicates significant increase; +, increase; −, no change; and =, significant decrease.

The authors would like to thank S. Meakins and Dr L. Manning for excellent technical assistance. We thank VICAL for generously providing us with the vector pkCMVint.polylinker. We thank Associate Prof I. Robertson for skilled assistance in statistical analyses.

Prepublished online as Blood First Edition Paper, November 21, 2002; DOI 10.1182/blood-2002-05-1521.

Supported by National Health and Medical Research Council, Australia (project grant no. 990393), the Cancer Foundation of Western Australia, and the Medical Research Foundation of Royal Perth Hospital.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Bennett
JM
Catovsky
D
Daniel
MT
et al
Proposals for the classification of the acute leukemias: French-American-British (FAB) co-operative group.
Br J Haematol.
33
1976
451
458
2
Klinken
SP
Nicola
NA
Johnson
GR
In vitro-derived leukemic cell lines induced by a raf and myc containing retrovirus differentiates in response to erythropoietin.
Proc Natl Acad Sci U S A.
85
1988
8506
8510
3
Farr
TJ
Lai
CM
Beilharz
MW
et al
A rapid fatal erythroleukemia caused by J2E cells can be treated ex vivo with erythropoietin.
Leukemia.
9
1995
900
907
4
Busfield
SJ
Klinken
SP
Erythropoietin-induced stimulation of differentiation and proliferation in J2E cells is not mimicked by chemical inductions.
Blood.
80
1992
412
419
5
Tilbrook
PA
Bittorf
T
Busfield
SJ
Chappell
D
Klinken
SP
Disrupted signalling in a mutant J2E cell line that shows enhanced viability, but does not proliferate or differentiate with erythropoietin.
J Biol Chem.
271
1996
3453
3459
6
Tilbrook
PA
Palmer
GA
Bittorf
T
et al
Maturation of erythroid cells and erythroleukemia development are affected by the kinase activity of Lyn.
Cancer Res.
61
2001
2453
2458
7
Tilbrook
PA
Colley
SM
McCarthy
DJ
Marias
R
Klinken
SP
Erythropoietin-stimulated Raf-1 tyrosine phosphorylation is associated with the tyrosine kinase Lyn in J2E erythroleukemic cells.
Arch Biochem Biophys.
396
2001
128
132
8
Baron
S
Tyring
SK
Flesichmann
WR
et al
The interferons: mechanisms of action and clinical applications.
JAMA
66
1991
1375
1383
9
Lublin
FD
Whitaker
JN
Eidelman
BH
Miller
AE
Arnason
BG
Burks
JS
Management of patients receiving interferon beta-1b for multiple sclerosis: report of a consensus conference.
Neurology.
46
1996
12
18
10
DeWit
R
Schattenkerk
JK
Boucher
CA
Bakker
PJ
Veenhof
KH
Donner
SA
Clinical and virological effect of high dose recombinant interferon alpha in disseminated AIDS-related Kaposis's sarcoma.
Lancet.
2
1988
1214
1216
11
Platanias
LC
Golomb
HM
Clinical use of interferons: hairy cell, chronic myelogenous and other leukemias.
IFN: Principles and Medical Applications.
Baron
S
Coppenhaver
FD
Fleishmann
WR
Jr
et al
1992
487
499
University of Texas Medical Branch at Galveston Press
Galveston, TX
12
Geng
Y
Yu
D
Blatt
LM
Taylor
MW
Gene therapy with an adeno-associated virus carrying an interferon gene results in tumor growth suppression and regression.
Cancer Gene Ther.
3
1996
31
38
13
Santodonato
L
Ferrantini
M
Gabriele
L
et al
Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon alpha-1 and herpes simplex thymidine kinase followed by ganciclovir.
Human Gene Therapy.
7
1996
1
10
14
Shamseddine
A
Taher
A
Jaafar
H
et al
Interferon alpha is an effective therapy for congenital dyserythropoietic anaemia type I.
Eur J Haematol.
65
2000
207
209
15
Foster
GR
Finter
NB
Are all type I human interferons equivalent?
J Viral Hepatitis.
5
1998
143
152
16
Seif
I
De Maeyer-Guignard
L
Structure and expression of a new murine IFN-α gene: MuIFN-αI9.
Gene.
43
1986
111
121
17
Isaacs
A
Lindenmann
J
Virus interference, 1: the interferon.
Proc R Soc Lond B.
147
1957
258
267
18
Gresser
I
Biological effects of interferons.
Soc Invest Dermatol.
95
1990
66
71
19
Fleischmann
CM
Fleishmann
WR
Jr
Effects of hyperthermia on the in vitro antiproliferative activities of HuIFN-α, HuIFN-β, and rHuIFN-γ employed separately and in combination.
J Biol Reg Homeostat Agents.
2
1988
173
185
20
Barber
GN
The interferons and cell death: guardians of the cell or accomplices of apoptosis.
Semin Cancer Oncol.
10
2000
103
111
21
Bartholome
EJ
Fabienne
W
Crusiaux
A
et al
IFNβ interferes with the differentiation of dendritic cells from peripheral blood mononuclear cells: selective inhibition of CD40-dependent IL-12 secretion.
J Interferon Cyt Res.
19
1999
471
478
22
Paquette
RL
Hsu
NC
Kiertscher
SM
et al
IFN-α and GM-CSF differentiate peripheral blood monocytes into potent antigen-presenting cells.
J Leukoc Biol.
64
1998
358
367
23
Luft
T
Pang
KC
Thomas
E
et al
Type I IFNs enhance the terminal differentiation of dendritic cells.
J Immunol.
161
1998
1947
1953
24
Radvanyi
LG
Banerjee
A
Weir
M
Messner
H
Low levels of IFN-α induce CD86 (B7.2) expression and accelerates dendritic cell maturation from peripheral blood mononuclear cells.
Scand J Immunol.
50
1999
499
509
25
Biron
CA
Interferons α and β as immune regulators—a new look.
Immunity.
14
2001
661
664
26
Tough
DF
Borrow
P
Sprent
J
Induction of bystander T cell proliferation by viruses and type I IFN in vivo.
Science.
272
1996
1947
1950
27
Brinkmann
V
Geiger
T
Alkan
S
Heusser
CH
IFN-α increases the frequency of IFN-γ-producing human CD4+ T cells.
J Exp Med.
178
1993
1655
1663
28
Estes
DM
Tuo
W
Brown
WC
Goin
J
Effects of type I/ type II IFNs and transforming growth factor-β on B-cell differentiation and proliferation: definition of costimulation and cytokine requirements for immunoglobulin synthesis and expression.
Immunol.
95
1998
604
611
29
Lutfalla
G
Holland
SJ
Cinato
E
et al
Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.
EMBO J.
14
1995
5100
5108
30
Uze
G
Lutfalla
G
Gresser
I
Genetic transfer of a functional human interferon alpha receptor into mouse cells: cloning and expression of its cDNA.
Cell.
60
1990
225
234
31
Colamonici
O
Yan
H
Domanski
P
et al
Direct binding to and tyrosine phosphorylation of the alpha subunit of the type I interferon receptor by p135tyk2 tyrosine kinase.
Mol Cell Biol.
14
1994
8133
8142
32
Domanski
P
Fish
E
Nadeau
OW
et al
A region of the beta subunit of the interferon alpha receptor different from box 1 interacts with Jak1 and is sufficient to activate the Jak-STAT pathway and induce an antiviral state.
J Biol Chem.
272
1997
26388
26393
33
Lehtonen
A
Matikainen
S
Julkunen
I
Interferons up-regulate STAT1, STAT2, and IRF family transcription factor gene expression in human peripheral blood mononuclear cells and macrophages.
J Immunol.
159
1997
794
803
34
Cho
SS
Bacon
CM
Sudarshan
C
et al
Activation of STAT4 by IL-12 and IFN-alpha: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation.
J Immunol.
157
1996
4781
4789
35
Fasler-Kan
E
Pansky
A
Wiederkehr
M
Battegay
M
Heim
MH
Interferon-alpha activates signal transducers and activators of transcription 5 and 6 in Daudi cells.
Eur J Biochem.
254
1998
514
519
36
Matikainen
S
Sareneva
T
Ronni
T
Lehtonen
A
Koskinen
PJ
Julkunen
I
Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2R alpha, c-myc, and pim-1 genes in human T cells.
Blood.
93
1999
1980
1991
37
Farrar
JD
Smith
JD
Murphy
TL
Leung
S
Stark
GR
Murphy
KM
Selective loss of type I interferon-induced STAT4 activation caused by a minisatellite insertion in Stat2.
Nature Immunol.
1
2000
65
69
38
Krishnan
K
Singh
B
Krolewski
JJ
Identification of amino acid residues critical for the Src-homology 2 domain-dependent docking of STAT2 to the interferon alpha receptor.
J Biol Chem.
273
1998
19495
19501
39
Stark
GR
Kerr
IM
Williams
BR
Silverman
RH
Schreiber
RD
How cells respond to interferons.
Annu Rev Biochem.
67
1998
227
264
40
Darnell
JE
STATs and gene regulation.
Science.
277
1997
1630
1635
41
Goh
KC
Haque
SJ
Williams
BR
p38 MAP kinase is required for STAT1 serine phosphorylation and transcriptional activation induced by interferons.
EMBO J.
18
1999
5601
5608
42
Uddin
S
Majchrzak
B
Woodson
J
et al
Activation of the p38 mitogen-activated protein kinase by type I interferons.
J Biol Chem.
274
1999
30127
30131
43
Uddin
S
Lekmine
F
Sharma
N
et al
The Rac1/p38 mitogen-activated protein kinase pathway is required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins.
J Biol Chem.
275
2000
27634
27640
44
Caraglia
M
Abbruzzese
A
Leardi
A
et al
Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.
Cell Death Differ.
6
1999
773
780
45
Arora
T
Floyd-Smith
G
Espy
M.J
Jelinek
DF
Dissociation between IFN-alpha-induced anti-viral and growth signaling pathways.
J Immunol.
162
1999
3289
3297
46
Swaminathan
N
Lai
CM
Beilharz
MW
Boyer
SJ
Klinken
SP
Biological activities of recombinant murine interferons alpha 1 and alpha 4: large difference in antiproliferative effect.
Antiviral Res.
19
1992
149
159
47
Lai
CM
Swaminathan
N
Beilharz
MW
Papadimitriou
J
Klinken
SP
Interferon-alpha inhibits erythropoietin-induced proliferation, but not differentiation, and restricts erythroleukemia development.
J Interferon Cytokine Res.
15
1995
669
675
48
Cooper
MC
Levy
J
Cantor
LN
Marks
PA
Rifkind
RA
The effect of erythropoietin on colonial growth of erythroid precursors in vitro.
Proc Natl Acad Sci U S A.
71
1974
1677
1680
49
Chappell
D
Tilbrook
PA
Bittorf
T
Colley
SM
Meyer
GT
Klinken
SP
Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases.
Cell Death Diff.
4
1997
105
113
50
Bowtell
DD
Rapid isolation of eukaryotic DNA.
Anal Biochem.
162
1987
463
465
51
Cull
VS
Bartlett
EJ
James
CM
Type I interferon gene therapy protects against cytomegalovirus-induced myocarditis.
Immunology.
106
2002
428
437
52
Chen
C
Okayama
H
High efficiency transformation of mammalian cells by plasmid DNA.
Mol Cell Biol.
7
1987
2745
2752
53
Lawson
CM
Yeow
W-S
Lee
CM
Beilharz
MW
In vivo expression of an interferon alpha gene by intramuscular injection of naked DNA.
J Interferon Cytokine Res.
17
1997
255
261
54
Haq
R
Halupa
A
Beattie
BK
Mason
JM
Zanke
BW
Barber
DL
Regulation of erythropoietin-induced STAT serine phosphorylation by distinct mitogen-activated protein kinases.
J Biol Chem.
277
2002
17359
17366
55
Gregory
RC
Jiang
N
Todokoro
K
Crouse
J
Pacifici
RE
Wojchowski
DM
Erythropoietin receptor and STAT5-specific pathways promote SKT6 cell hemoglobinization.
Blood.
92
1998
1104
1118
56
Socolovsky
M
Fallon
AE
Wang
S
Brugnara
C
Lodish
H
Fetal anaemia and apoptosis of red cell progenitors in Stat5a-/- 5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction.
Cell.
98
1999
181
191
57
Cull
VS
Tilbrook
PA
Adenan
AS
et al
Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia in vivo.
Oncogene.
19
2001
953
960
58
Bartlett
EJ
Cull
VS
Brekalo
NL
Lenzo
JC
James
CM
Synergy of IFN-A6 and IFN-B DNA immunotherapy for cytomegalovirus infection.
Immunol Cell Biol.
80
2002
425
435
59
Harle
P
Cull
VS
Agbaga
M-P
et al
Differential effect of murine type I IFN transgene in antagonizing HSV-1 replication.
J Virol.
66
2002
6558
6567
60
Harle
P
Cull
V
Guo
L
Papin
J
Lawson
C
Carr
D
Transient transfection of mouse fibroblasts with type I interferon transgenes provides various degrees of protection against herpes simplex virus infection.
Antiviral Res.
56
2002
39
49
61
Borden
EC
Lindner
D
Dreicer
R
Hussein
M
Peereboom
D
Second-generation interferons for cancer: clinical targets.
Semin Cancer Biol.
10
2000
125
144
62
Geng
Y
Yu
D
Blatt
LM
Taylor
MW
Tumor suppressor activity of the human consensus type I interferon gene.
Cytokines Mol Ther.
1
1995
289
300
63
Menike
D
Wickramasinghe
SN
Effects of four species of interferon-α on cultured erythroid progenitors from congenital dyserythropoietic anaemia type I.
Brit J Haematol.
103
1998
825
830
64
Busfield
SJ
Riches
KJ
Sainsbury
AJ
Garcia-Webb
P
Klinken
SP
Retrovirally-produced erythropoietin effectively induces differentiation and proliferation of J2E erythroid cells.
Growth Factors.
9
1993
87
97
65
Callus
B
Tilbrook
PA
Busfield
SJ
Cull
VS
Bittorf
T
Klinken
SP
Amiloride suppresses erythropoietin-induced proliferation and MAP kinase, but potentiates differentiation of J2E cells.
Exp Cell Res.
219
1995
39
46
66
Busfield
SJ
Spadaccini
A
Riches
KJ
Tilbrook
PA
Klinken
SP
The major erythroid DNA-binding protein GATA-1 is stimulated by erythropoietin but not by chemical inducers of erythroid differentiation.
Eur J Biochem.
230
1995
475
480
67
Choi
I
Muta
K
Wickrema
A
Krantz
SB
Nishimura
J
Nawata
H
Interferon gamma delays apoptosis of mature erythroid progenitor cells in the absence of erythropoietin.
Blood.
95
2000
3742
3749
68
Anderson
CC
Matzinger
P
Danger: the view from the bottom of the cliff.
Sem Immunol.
12
2000
231
238
69
Tarumi
T
Sawada
K
Sato
N
et al
Interferon-alpha-induced apoptosis in human erythroid progenitor cells.
Exp Hematol.
23
1985
1310
1318
70
Roos
G
Leanderson
T
Lundgren
E
Interferon-induced cell cycle changes in human hematopoietic cell lines and fresh leukemic cells.
Cancer Res.
44
1984
2358
2362
71
Sangfelt
O
Erickson
S
Castro
J
Heiden
T
Einhorn
S
Grander
D
Induction of apoptosis and inhibition of cell growth are independent responses to interferon-alpha in hematopoietic cell lines.
Cell Growth Differ.
8
1997
343
352
72
Grumbach
IM
Fish
EN
Uddin
S
et al
Activation of the Jak-STAT pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha.
J Interferon Cytokine Res.
19
1999
797
801
73
Fish
EN
Uddin
S
Korkmaz
M
Majchrzak
B
Druker
BJ
Platanias
LC
Activation of a CrkL-STAT5 signaling complex by type I interferons.
J Biol Chem.
274
1999
571
573
74
Stancato
LF
Sakatsume
M
David
M
et al
Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway.
Mol Cell Biol.
17
1997
3833
3840
75
Berger
LC
Tamir
A
Ben-David
Y
Hawley
RG
Dose-dependent activation of p21ras by IFN-beta.
J Interferon Cytokine Res.
17
1997
757
762
76
Burfoot
MS
Rogers
NC
Watling
D
et al
Janus kinase-dependent activation of insulin receptor substrate 1 in response to IL-4, oncostatin M, and the interferons.
J Biol Chem.
272
1997
24183
24190
77
Rani
MR
Leaman
DW
Han
Y
et al
Catalytically active TYK2 is essential for interferon-beta-mediated phosphorylation of STAT3 and interferon-alpha receptor-1 (IFNAR-1) but not for activation of phosphoinositol 3-kinase.
J Biol Chem.
274
1999
32507
32511
78
Lutfalla
G
Holland
SJ
Cinato
E
et al
Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.
EMBO J.
14
1995
5100
5108
79
Platanias
LC
Uddin
S
Domanski
P
Colamonici
OR
Differences in interferon alpha and beta signaling: interferon beta selectively induces the interaction of the alpha and betaL subunits of the type I interferon receptor.
J Biol Chem.
271
1996
23630
23633
80
David
M
Chen
HE
Goelz
S
Larner
AC
Neel
BG
Differential regulation of the alpha/beta interferon-stimulated Jak/STAT pathway by the SH2 domain-containing tyrosine phosphatase SHPTP1.
Mol Cell Biol.
15
1995
7050
7058
81
Hilkens
CMU
Schlaak
JF
Kerr
IM
Molecular analysis of responses to interferon-alpha subtypes in human T cells and dendritic cells.
J Interferon Cytokine Res.
22(suppl 1)
2002
S56
82
Cutrone
EC
Langer
JA
Contributions of cloned type I interferon receptor subunits to differential ligand binding.
FEBS Lett.
404
1997
197
202
83
Lewerenz
M
Mogensen
KE
Uze
G
Shared receptor components but distinct complexes for α and β-interferons.
J Mol Biol.
282
1998
585
599
84
Chunthrapai
A
Gibbs
V
Lu
J
et al
Determination of residues involved in ligand binding and signal transmission in human IFN-α receptor 2.
J Immunol.
163
1999
766
773
85
Williams
BR
Signal integration via PKR.
Sci STKE.
89
2001
RE2
Review.
86
Kuhen
KL
Samuel
CE
Mechanisms of interferon action: functional characterization of positive and negative regulatory domains that modulate transcriptional activation of the human RNA-dependent protein kinase Pkr promoter.
Virology.
254
1999
182
195
87
Landolfo
S
Gribaudo
G
Angeretti
A
Gariglio
M
Mechanisms of viral inhibition by interferons.
Pharmacol Therapeutics.
65
1995
415
442
88
Meurs
E
Chong
K
Galabru
J
et al
Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.
Cell.
62
1990
379
390
89
Kumar
A
Yang
YL
Flati
V
et al
Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.
EMBO J.
16
1997
406
416
90
Wong
LH
Krauer
KG
Hatzinisiriou
I
et al
Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma.
J Biol Chem.
272
1997
28779
28785
91
Raveh
T
Hovanessian
AG
Meurs
EF
Sonenberg
N
Kimchi
A
Double-stranded RNA-dependent protein kinase mediates c-Myc suppression induced by type I interferons.
J Biol Chem.
271
1996
25479
25484
92
Salzberg
S
Heller
A
Zou
JP
Collart
FR
Huberman
E
Interferon-independent activation of (2′-5′) oligoadenylate synthetase in Friend erythroleukemia cell variants exposed to HMBA.
J Cell Sci.
109
1996
1517
1526
93
Cull
VS
Broomfield
S
Bartlett
EJ
Brekalo
NL
James
CM
Coimmunisation with type I IFN genes enhances protective immunity against cytomegalovirus and myocarditis in gB DNA-vaccinated mice.
Gene Therapy.
9
2002
1369
1378
94
Li
S
Xia
X
Zhang
X
Suen
J
Regression of tumours by IFN-alpha electroporation gene therapy and analysis of the responsible genes by cDNA array.
Gene Therapy.
9
2002
390
397
95
Hiroishi
K
Tuting
T
Lotze
MT
IFN-alpha-expressing tumor cells enhance generation and promote survival of tumor-specific CTLs.
J Immunol.
164
2000
567
572
96
Currier
NL
Miller
SC
Influence of an interferon inducer on bone marrow transplant reconstitution in irradiated, leukemic mice: elevated natural killer cell numbers and improved life span.
Nat Immunol.
16
1998
6
17
97
Gresser
I
Belardelli
F
Endogenous type I interferons as a defence against tumours.
Cytokine Growth Factor Rev.
13
2002
111
118
98
Piehler
J
Rosiman
LC
Shreiber
G
New structural and functional aspects of the type I interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface.
J Biol Chem.
275
2000
40425
40433

Author notes

Cassandra James, Division of Veterinary and Biomedical Sciences, Murdoch University, South St, Murdoch 6150, Western Australia, Australia; e-mail:casjames@central.murdoch.edu.au.

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