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The Antineoplastic Agent Bryostatin-1 Differentially Regulates IFN- Receptor Subunits in Monocytic Cells: Transcriptional and Posttranscriptional Control of IFN-R2¹

Carmen S. Garcia^2,*, Rafael E. Curiel^2,*, James M. Mwatibo^*, Sidney Pestka, Huifen Li and Igor Espinoza-Delgado^3,

^* Department of Medicine and Stanley S. Scott Cancer Center, Louisiana State University Medical Center, New Orleans, LA 70112; Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854; and Hematology-Oncology Section, Gerontology Research Center, National Institute on Aging, Baltimore, MD 21224

	Abstract

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Bryostatin-1 (Bryo-1) is a potent ligand and modulator of protein kinase C that exerts antineoplastic and immunomodulatory activities both in vitro and in vivo. We have previously reported that Bryo-1 synergized with IFN- to induce NO synthase and NO by macrophages. To determine whether this effect was associated with changes in levels of IFN-R, we investigated the effects of Bryo-1 on the expression and regulation of IFN-R chains in monocytic cells. Northern blot analysis revealed that Bryo-1 treatment of the human monocytic cell lines MonoMac6 and THP-1 and human monocytes enhanced the expression of IFN-R2 mRNA but did not affect IFN-R1 mRNA expression. Bryo-1 increased IFN-R2 mRNA in a dose-dependent manner as early as 3 h posttreatment. Bryo-1-induced up-regulation of IFN-R2 mRNA levels is not dependent on de novo protein synthesis as shown by cell treatment with the protein-synthesis inhibitor cycloheximide. Bryo-1 treatment increased the IFN-R2 mRNA half-life by 2 h. EMSA analysis from Bryo-1-treated MonoMac6 cells showed an increased nuclear protein binding to the NF-B motif present in the 5' flanking region of the human IFN-R2 promoter that was markedly decreased by pretreatment with the NF-B inhibitor SN50. These results show for the first time that Bryo-1 up-regulates IFN-R2 expression in monocytic cells. Given the pivotal role that IFN- exerts on monocyte activation and in the initiation and outcome of the immune response, the induction of IFN-R2 by Bryo-1 has significant implications in immunomodulation and could overcome some of the immune defects observed in cancer patients.

	Introduction

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Bryostatin-1 (Bryo-1),⁴ a naturally occurring macrocyclic lactone and a potent ligand and modulator of protein kinase C (PKC) (1, 2), has a broad spectrum of biological effects both in vitro and in vivo, and it has been shown to have both antineoplastic and immunomodulatory effects. Bryo-1 has significant direct antitumor activity against a variety of murine and human tumor cell lines (3). In addition to its direct antineoplastic activity, Bryo-1 is also a powerful activator and modulator of host immune effector cells, which may indirectly result in inhibition of a tumor’s growth. Bryo-1 induces the proliferation and activation of human T and B cells (4, 5), triggers the development of tumor-specific CTL that can traffic and mediate tumor regression (6), and costimulates T lymphocytes to produce a variety of cytokines, including IFN- and IL-2 (7), both of which display antitumor effects.

IFN-, an important immunomodulatory cytokine secreted by activated T lymphocytes and NK cells (8), is a potent activator of human monocytic cells. IFN- activates and differentiates human monocytes (9), leading to an increase in the expression of MHC class I and class II molecules (8), tumor-associated Ags, transferrin receptor expression (10), tumoricidal activity (9), and regulation of cytokine receptors (9, 11). In addition, others and we (12, 13) have also shown that IFN- increases the expression of the costimulatory molecule B7-2 in monocytic cells and in primary human monocytes. IFN- is known to signal through a unique receptor complex (14) ubiquitously expressed on all nucleated cells, which comprises two different subunits: IFN-R1 chain (IFN-R1) and IFN-R2 chain (IFN-R2) (15, 16). IFN-R1 is a species-specific cell surface receptor (17, 18) encoded on human chromosome 6 (19) and is the binding subunit of the IFN-R complex. IFN-R1 is important for both receptor-ligand internalization and cell signaling (20, 21). IFN-R2, localized to human chromosome 21 (16), is essential to initiate the signal transduction cascade in response to IFN- as demonstrated by cotransfection studies where only cells expressing both IFN-R subunits were able to respond to IFN- treatment (22, 23). Each chain of the IFN-R is constitutively associated with a specific JAK: IFN-R1 with JAK1 and IFN-R2 with JAK2 (24). Both IFN-R subunits are preassociated at the cell surface in unstimulated cells (25). After addition of ligand, the intracellular domains move apart (25), and signal transduction begins through phosphorylation of Jak1, Jak2, and Stat1 (14, 22, 24).

We have previously demonstrated that IFN- synergizes with Bryo-1 for the expression of inducible NO synthase mRNA and NO by macrophages (26). Based on these results, we hypothesized that one of the potential mechanisms responsible for the synergistic effect of Bryo-1 and IFN- is the up-regulation of the IFN-R subunits by Bryo-1. To investigate this hypothesis, we studied the expression and regulation of IFN-R mRNA in the human monocytic cell line MonoMac6 (MM6) in response to Bryo-1. MM6 cells have been extensively characterized, and they display phenotypic and functional features of mature human monocytes (27), including enhanced Ag expression in response to IFN-. In the present study, we demonstrated for the first time that Bryo-1 differentially regulates the IFN-R subunits. Bryo-1 enhances IFN-R2 mRNA accumulation in human monocytic cells but does not affect IFN-R1 mRNA expression. Up-regulation of IFN-R2 expression by Bryo-1 occurs rapidly and through mechanisms that do not require new protein synthesis. We also provide first-time evidence that the enhancement of IFN-R2 mRNA expression by Bryo-1 is controlled through both transcriptional and posttranscriptional mechanisms.

The transcription NF, NF-B, has been implicated in the control of diverse cellular processes, including transformation, induction of proliferation, suppression of apoptosis (28), modulation of the antiviral response (IFN- and IFN-), induction of inflammatory cytokines such as IL-1 and TNF- (29), as well as chemokines, and cell invasion and angiogenesis in oncogenesis (30). Wang et al. (31) reported that PKC activators, including Bryo-1, induced TNFR-associated factor 1 mRNA expression in human colon cancer cells through a Ca²⁺-dependent PKC/Raf-1/ERK/NFB-dependent pathway. Based on previous analysis of the promoter region of the human IFN-R2 gene identifying a KB site that matched a NF-B-like sequence found in the mouse IFN-R2 promoter (32), we assessed whether this NF-B site was a potential Bryo-1-responsive transcriptional region in monocytic cells.

EMSA analysis revealed that Bryo-1 treatment enhanced the binding of the NF NF-B to its NF-B motif present in the 5' flanking region of the human IFN-R2 promoter. Furthermore, NF-B inhibitors block the enhanced expression of IFN-R2 mRNA, suggesting that NF-B may be involved in the transcriptional regulation of IFN-R2 by Bryo-1. Overall, these findings constitute the first report of an antineoplastic agent controlling the expression of the IFN-R2, a key component to all IFN--mediated immunomodulatory activities, in monocytic cells. We also determined the molecular mechanisms responsible for the enhancement of the IFN-R2 gene expression.

	Materials and Methods

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Cell culture conditions

MM6 and THP-1, two human monocytic cell lines, were obtained from the repository of the National Cancer Institute, Frederick Cancer Research and Development Center, and cultured as described previously (27). Briefly, MM6 cells were cultured at 37°C in a 5% CO₂-humidified atmosphere in DMEM (BioWhittaker) supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, and 20 mM HEPES (Invitrogen Life Technologies), hereafter referred to as complete culture medium, and 15% heat-inactivated FCS (HyClone Laboratories). THP-1 cells were cultured under the same conditions in RPMI 1640 (BioWhittaker) complete culture medium supplemented with 10% heat-inactivated FCS (HyClone Laboratories). Peripheral blood leukocytes were obtained from normal healthy volunteers by leukapheresis using a Fenwell CS-3000 blood cell separator (Fenwell Laboratories). The ethical review board of the Louisiana State University Health Sciences Center approved the protocol, and all patients signed a written informed consent before treatment. Mononuclear cells were separated by density gradient centrifugation on Ficoll-Paque (Amersham Biosciences), and monocytes were then purified in suspension from the unfractionated mononuclear leukocyte preparation by countercurrent centrifugal elutriation in a Beckman JE-6 elutriation chamber and rotor system (Beckman Instruments) as described elsewhere (33). The purity of monocyte preparations was 94 ± 3%, as assessed by morphology on Giemsa-stained cytocentrifuge slide preparations and by flow cytometry using the monocyte-specific mAb to CD14 (BD Pharmingen). Viability, as determined by the trypan blue exclusion test, was >99%. Monocytes were cultured at 37°C in a 5% CO₂-humidified atmosphere in RPMI 1640 (BioWhittaker) complete culture medium supplemented with 10% heat-inactivated FCS (HyClone Laboratories).

Cell treatment

Exponentially growing MM6 and THP-1 cells were plated in 100-mm² tissue culture plates (Corning Glass Works) at a density of 5 x 10⁵ cells/ml in fresh complete medium or medium supplemented with different concentrations of Bryo-1. Clinical grade Bryo-1 (Bryo-1 conversion factor: 1.0 ng/ml = 0.904 nmol/L) was a gift from A. Fallavollita, Jr. (Cancer Therapy Evaluation Program, Division of Cancer Treatment, Diagnosis, and Centers, National Cancer Institute, National Institutes of Health). Cells were harvested at the indicated time points and were used for total RNA extraction and nuclear cell lysate preparations as described below. In other experiments, MM6 cells were treated with Bryo-1 along with 5 µg/ml actinomycin D (Act D; Sigma-Aldrich) or 10 µg/ml cycloheximide (CHX) (Sigma-Aldrich) to inhibit cellular RNA and protein synthesis, respectively. Monocytes were cultured in 15-cm Lux plates (Miles Scientific) at 2 x 10⁶ cells/ml in medium alone or supplemented with various concentrations of Bryo-1. SN50, a specific inhibitor of nuclear translocation of NF-B, and the inactive control peptide inhibitor NF-B SN50M (Calbiochem) were solubilized in PBS.

Northern blot analysis

MM6 cells were cultured in medium alone or with Bryo-1. Cells were harvested and lysed with TRIzol (Invitrogen Life Technologies), and total RNA was extracted and purified according to the manufacturer’s specifications. Standard Northern blot analysis was performed as described previously (33). Briefly, 20 µg of total RNA of each sample was electrophoresed under denaturing conditions, blotted onto nytran membranes (Schleicher & Schuell), and cross-linked by UV irradiation. Membranes were prehybridized at 42°C in Hybrisol (Oncor) and hybridized overnight with 2 x 10⁶ cpm/ml -³²P labeled probe. Membranes were then washed three times at room temperature for 10 min in 2x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate (pH 7.0)), containing 0.1% SDS, and twice at 65°C for 20 min in 0.2x SSC and 0.1% SDS before being autoradiographed using Kodak Biomax-MR films (Eastman Kodak) and intensifying screens at –70°C. The human cDNA IFN-R1 (18), IFN-R2 (16), and the human GAPDH (BD Clontech) cDNAs were labeled by random priming and [-³²P]dCTP (3000 Ci/mmol; NEN Life Science Products) and purified using ProbeQuant G-50 micro columns (Amersham Biosciences). The intensity of the bands from the autoradiograms was then quantified using an AlphaImager 2000 (Alpha Innotech). The graphs were generated from the intensity of the IFN-R2 or IFN-R1 mRNAs and normalized to GAPDH mRNA.

Nuclear run-on transcription assay

Nuclear run-on experiments were performed using a previously described protocol (9). Cells (50 x 10⁶ cells/sample) were lysed, and nuclei were isolated in 4 ml of lysis buffer (10 mM Tris-HCl (pH 7.4), 3 mM MgCl₂, 10 mM NaCl, 150 mM sucrose, and 0.5% Nonidet P-40 (Sigma-Aldrich)) for 5 min on ice. The nuclei were spun at 167 x g for 5 min at 4°C, and pellets were resuspended in lysis buffer without Nonidet P-40. The nuclei were pelleted again and resuspended in 150 µl of freezing buffer (50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl₂, and 0.1 mM EDTA). Run-on assays were performed by adding 150 µl of 2x transcription buffer (20 mM Tris-HCl (pH 8.0), 300 mM KCl, 10 mM MgCl₂, 200 mM sucrose, 20% glycerol, 1 mM DTT, and 0.5 mM of each ATP, GTP, and CTP) and 100 µCi of 800 Ci/mmol [³²P]uridine triphosphate (NEN Life Science Products) to 150 µl of nuclei suspension. After samples were incubated at 29°C for 30 min, 30 µl of 200 mM CaCl₂ and 30 µl of 1 U/ml RNase-free DNase 1 (Promega) were added to each reaction and further incubated for 10 min at 29°C. Labeled transcripts were isolated using TRIzol (Invitrogen Life Technologies) and purified according to the manufacturer’s specifications. Equal amounts of radioactivity (2 x 10⁶ cpm of labeled RNA) were added in 2 ml of Hybrizol (Oncor) to nytran membranes on which 1 µg of denatured full-length human IFN-R2 cDNA (1.8 kb) and chicken -actin cDNA (1.8 kb, HindIII fragment; Oncor) were immobilized using a slot blot apparatus (Invitrogen Life Technologies) and a UV cross-linker (Fisher Scientific). Hybridization was conducted at 42°C for 48 h. Filters were washed three times at 42°C for 15 min with 2x SSC/0.1% SDS and twice at 65°C for 20 min with 0.2x SSC/0.1% SDS. Filters were then autoradiographed at –70°C. Data were normalized for the content of -actin present in each sample using an AlphaImager 2000 (Alpha Innotech).

mRNA half-life determination

Following incubation of MM6 cells in medium alone or medium containing 1.0 ng/ml Bryo-1 for 6 h, Act D was added to the cultures to a final concentration of 5 µg/ml. The cells were harvested at the indicated lengths of time through 8 h post-Act-D treatment, and the levels of IFN-R2 mRNA were quantified by standard Northern blot analysis. A graph was generated using the integrated values of the bands from the autoradiograms of the IFN-R2 mRNA remaining after Act D treatment, as determined by densitometry, and normalized to the respective values of GAPDH mRNA.

EMSA

Double-stranded oligonucleotide containing a potential NF-B-binding transcription factor-like motif from the 5'-flanking region of human IFN-R2 promoter (34) (5'-GAGCGGGAAAGTCCCGCGCGG-3') located at position –221 to –230 was end-labeled with [-³²P]dCTP (3000 Ci/mmol; NEN Life Science Products) using the Klenow fragment of DNA polymerase I (Amersham Biosciences). Labeled DNA probe was purified using ProbeQuant G-50 microcolumns (Amersham Biosciences), adjusted to 20,000 cpm/µl, and stored at –70°C until use. EMSA was performed as described previously (35). For supershift analysis, nuclear extracts were incubated with 1 µl (200 ng) of polyclonal Abs to NF-B p50 (H-119)X, NF-B p65 (A)X, or c-Rel (N-466)X (Santa Cruz Biotechnology) for 30 min at room temperature before the addition of ³²P-labeled DNA probe (50,000 cpm). Cold competition experiments were performed by including unlabeled probes for the NF-B-like motif (5'-GAGCGGGAAAGTCCCGCGCGG-3') or the human IL-2 distal NFAT site (5'-AAAGAAAGGAGGAAAAACTGTTTCATACAG-3'). Results were visualized by autoradiography after 6–12 h exposure using Kodak Biomax-MR films (Eastman Kodak) and intensifying screens at –70°C.

Real-time RT-PCR

Monocytes were cultured for 12 h at 1.5 x 10⁶ cells/ml. Cells were pretreated with NF-B SN 50 or the inactive control peptide NF-B SN 50M for 30 min and then 0.01 ng/ml Bryo-1 was added for 12 h. Cells were then lysed in TRIzol, and total RNA was purified according to the manufacturer’s instructions. First-strand cDNA synthesis was conducted in a total volume of 100 µl containing 1 µg of total RNA with TaqMan reverse transcription kit using random primer according to the manufacturer’s protocol (TaqMan; Applied Biosystems). The cDNA was amplified using primers described previously (36): IFN-R2, 5'-TGGACAAGGACAGCTCACCA-3' and 5'-TCAAAGCGTTTGGAGAACATCTT-3'; and GAPDH, 5'-GTTCGACAGTCAGCCGCATC-3' and 5'-GGAATTTGCCATGGGTGGA-3'. Real-time quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. The reactions were performed using ABI PRISM 7700 Sequence Detector. The condition for target sequences amplification was as follows: the initial step of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C as described previously (36). All samples were run in triplicate. The relative amounts of IFN-R2 transcripts were normalized to GAPDH, and the difference fold among samples was calculated as described in detail in ABI PRISM Sequence Detection System User Bulletin 2 (PE Applied Biosystems).

	Results

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Bryo-1 enhances IFN-R2 mRNA expression in human monocytic cells

To determine whether MM6 cells responded to Bryo-1 with changes in IFN-R mRNA expression, MM6 were cultured for 6 h in medium alone or in the presence of 1 ng/ml Bryo-1, a dose previously shown to induce activation of monocytes and macrophages (26, 33). Total RNA was extracted and Northern blot analysis was performed. As shown in Fig. 1A, IFN-R2 mRNA was constitutively expressed in the medium-treated cells, and Bryo-1 treatment of MM6 cells led to a significant enhanced expression of IFN-R2 mRNA (4.5-fold induction over medium-treated cells). Noteworthy, Bryo-1 treatment of MM6 cells did not affect the basal expression of IFN-R1 mRNA (Fig. 1B). As previously reported (16), the IFN-R2 cDNA hybridized to the major transcript for the human IFN-R2 of 1.8 Kb and also to minor transcripts of larger size. To rule out the possibility that Bryo-1 could up-regulate the expression of IFN-R1 mRNA at a later time point, we studied the kinetics of IFN-R1 and IFN-R2 expression in MM6 cells in response to 1 ng/ml Bryo-1. As seen in Fig. 2A, the constitutive expression of IFN-R1 mRNA was not significantly altered by Bryo-1 treatment at any of the time points tested. On the other hand, an early enhanced expression of the IFN-R2 mRNA was observed within 3 h (2-fold increase) after stimulation with Bryo-1, and maximal increase was noticed from 6 to 12 h (4- to 4.5-fold increases); by 24 h, following Bryo-1 treatment, IFN-R2 mRNA expression started to decline (Fig. 2B). Dose-response experiments were performed to determine the optimal concentration of Bryo-1 needed to induce maximal enhanced expression of IFN-R2 mRNA. MM6 cells were cultured in medium alone or in the presence of increasing concentrations of Bryo-1. After 6 h of stimulation, total RNA was extracted and analyzed by Northern blot for IFN-R2 mRNA expression. As seen in Fig. 3, Bryo-1 induced a dose-dependent increase of IFN-R2 mRNA. A concentration of 0.1 ng/ml Bryo-1 was sufficient to induce a minor increase in IFN-R2 mRNA levels, and a steady augmentation of IFN-R2 transcript expression was observed with higher doses of Bryo-1 up to 10.0 ng/ml (8-fold increase). Bryo-1 at the concentrations used in the present study was not toxic to either monocytic cell lines or human monocytes as assessed by trypan blue exclusion (data not shown). Since 1.0 ng/ml Bryo-1 is potentially an achievable dose (37) and augmented IFN-R2 mRNA expression by 5-fold, this dose was used in all subsequent experiments. To discern whether the enhanced IFN-R2 mRNA expression was an isolated event only seen in MM6 cells, THP-1 cells and human monocytes were cultured for 18 and 12 h, respectively, in medium alone or in the presence of various concentrations of Bryo-1. As shown in Fig. 4A, stimulation of THP-1 cells with 1 ng/ml Bryo-1 resulted in a 5-fold increase in IFN-R2 mRNA expression over the medium control. Similarly, human monocytes treated with 0.01 ng/ml Bryo-1 had a significant increase in the expression of IFN-R2 mRNA (Fig. 4B). This dose of Bryo-1 has been previously shown to induce activation of human monocytes (33).

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Bryo-1 treatment of MM6 cells up-regulates IFN-R2 mRNA expression. MM6 cells were cultured for 6 h in medium alone or with 1.0 ng/ml Bryo-1. Total cellular RNA was extracted and analyzed by Northern blot for IFN-R2 mRNA expression (A). The same membrane was rehybridized with the IFN-R1 and GAPDH cDNA probes (B), respectively. C, The relative induction of IFN-R1 and IFN-R2 mRNAs after the intensity of the bands from autoradiograms were normalized to the GAPDH housekeeping gene control as indicated in Materials and Methods. Data shown are from one representative experiment of four performed.

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Kinetics of Bryo-1-induced up-regulation of IFN-R2 mRNA. A, MM6 cells were stimulated in the presence or absence of 1 ng/ml Bryo-1 for the indicated lengths of time. Total cellular RNA was isolated, and Northern blot analysis for IFN-R1 mRNA expression was performed. The same filter was subsequently probed with IFN-R2 cDNA and then with the GAPDH probe to ensure that comparable amounts of RNA were loaded in each lane. B, Quantitative analysis of IFN-R2 and IFN-R1 mRNA expression. The graph was generated as explained in the legend of Fig. 1. Data shown are from one representative experiment of three performed.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. IFN-R2 mRNA expression is enhanced in a dose-dependent manner by Bryo-1. MM6 cells were cultured for 6 h in the absence or presence of increasing concentrations of Bryo-1. Total cellular RNA was extracted and analyzed by Northern blot for IFN-R2 mRNA expression. The same membrane was rehybridized with GAPDH to control that equal amounts of RNA were loaded in each lane. Northern blot analysis for IFN-R2 expression (upper panel) and quantitative analysis of IFN-R2 mRNA expression (lower panel) are shown. As described in Materials and Methods, the intensity of the bands was normalized to the GAPDH housekeeping gene control, and the graph was generated with the relative values obtained after normalization. Data shown are from one representative experiment of two performed.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Bryo-1 treatment of THP-1 cells and human monocytes up-regulates IFN-R2 mRNA expression. A, THP-1 cells were cultured for 18 h in medium alone or with 1.0 ng/ml Bryo-1. Human monocytes were cultured for 12 h in medium alone or in the presence of 0.01 ng/ml Bryo-1 (B). Total cellular RNA was extracted at the indicated time points and analyzed by Northern blot for IFN-R2 mRNA expression. The same membranes were rehybridized with GAPDH probe to control that equal amounts of RNA were loaded in each lane, and a graph was generated with the relative values obtained after normalization (bottom panels). Data shown are from one representative experiment of two performed.

The transcriptional activity of the IFN-R2 gene is enhanced by treatment with Bryo-1

To investigate whether the increased expression of IFN-R2 mRNA by Bryo-1 involved changes in IFN-R2 gene transcription, nuclear run-on experiments were performed. MM6 cells were incubated with medium alone or supplemented with 1 ng/ml Bryo-1, the nuclei were isolated at 4 h after treatment, and in vitro transcription assays were performed. As seen in Fig. 5, the IFN-R2 gene was transcriptionally active in medium-treated cells and Bryo-1 treatment for 4 h further increased the rate of transcription of the IFN-R2 gene by 3-fold.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Bryo-1 augments IFN-R2 gene transcription. MM6 cells (5 x 107 cells/point) were treated with medium alone or with 1 ng/ml Bryo-1, and nuclei were isolated after 4 h. The rate of transcription of the IFN-R2 gene was then assessed by nuclear run-on analysis as described in Materials and Methods. The graph (lower panel) was generated as described in the legend of Fig. 1 with the relative values obtained after normalization of the intensity of the bands to the respective amounts of -actin mRNA. Data presented are from one of three similar experiments.

Bryo-1 enhances IFN-R2 mRNA stability

Experiments were performed to determine whether Bryo-1 affected the stability of IFN-R2 mRNA. MM6 cells were incubated for 6 h with medium alone or supplemented with 1 ng/ml Bryo-1. After the 6-h incubation period, Act-D was added to the cultures for the indicated lengths of time to block further RNA transcription. Northern blot analysis revealed that IFN-R2 mRNA decayed with different kinetics in cells cultured in medium alone and in Bryo-1-treated cells (Fig. 6). The level of IFN-R2 mRNA in medium-treated cells decreased by 50% (half-life (t_1/2)) after 2 h and 50 min, and IFN-R2 mRNA were reduced substantially after 6 h of Act D treatment. On the other hand, Bryo-1-treated cells displayed an enhanced IFN-R2 mRNA stability resulting in a t_1/2 of 4 h and 20 min, and IFN-R2 mRNA levels were detectable for at least 8 h after Act-D treatment. Similar results were observed with the monocytic cell line THP-1 where Bryo-1 treatment (1 ng/ml) increased IFN-R2 mRNA stability (t_1/2 = 4.2 h) by almost 3 h compared with medium-treated cells (t_1/2 = 1.3 h; data not shown).

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Treatment of MM6 cells with Bryo-1 increases IFN-R2 mRNA stability. MM6 cells were incubated for 6 h in medium alone or supplemented with 1 ng/ml Bryo-1. After 6 h, cells were treated with 5 µg/ml Act D, and their total cellular RNA was collected and analyzed by Northern blot for IFN-R2 mRNA expression at the indicated time points (upper panel). The graph (lower panel) was generated as described in Materials and Methods, and data are presented as the relative amounts of IFN-R2 mRNA remaining after adding Act D and normalized to the respective amounts of GAPDH. Data shown are from one representative experiment of two performed.

Protein synthesis is not required for the Bryo-1-induced up-regulation of IFN-R2 mRNA

To determine whether active protein synthesis was necessary for the Bryo-1 up-regulation of IFN-R2 mRNA, MM6 cells were incubated for 6 h with medium alone or supplemented with 1 ng/ml Bryo-1 in the absence or presence of the protein-synthesis inhibitor CHX. As shown in Fig. 7, the addition of CHX to Bryo-1-treated MM6 cells did not decrease the enhanced IFN-R2 mRNA expression. These results suggest that the Bryo-1-induced up-regulation of IFN-R2 mRNA expression is not dependent on de novo protein synthesis. Noteworthy, addition of CHX to medium-treated MM6 cells caused induction of the basal IFN-R2 mRNA expression.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. De novo protein synthesis is not required for the Bryo-1-enhanced expression of IFN-R2 mRNA. MM6 cells were incubated for 6 h in medium alone or supplemented with 1 ng/ml Bryo-1 and in the absence or presence of 10 µg/ml CHX. Total cellular RNA was extracted and analyzed by Northern blot for IFN-R2 mRNA expression (upper panel). The graph represents the quantitative analysis of IFN-R2 mRNA expression, and it was generated with the relative values obtained after normalization to the GAPDH housekeeping gene control (lower panel). Data shown are from one of two similar experiments.

Bryo-1 enhances NF-B binding in MM6 cells

Previous studies have demonstrated that the 5' regulatory sequence of the human IFN-R2 gene between nt –411 and –90 is important for promoter activity (34). Nucleotide sequence analysis of this region revealed a NF-B site localized from –221 to –230 that matched the previously reported NF-B-like site (GGAAAGTCCC) found in the mouse IFN-R2 promoter (32). To ascertain whether this NF-B site was a potential Bryo-1-responsive region, we performed EMSA analysis using this specific ³²P-labeled NF-B oligonucleotide. In the presence of nuclear extracts from medium-treated MM6 cells, constitutive NF-B complexes formation was observed (Fig. 8, lane 1, bands A and B), and Bryo-1 treatment (1 ng/ml) further enhanced protein complex binding to this DNA element (Fig. 8, lane 2, band A). To determine the binding specificity of these complexes, competition analysis with specific and nonspecific competitors were performed. As shown in Fig. 8, binding of the complexes to the DNA probe was specific because both the constitutive and Bryo-1-increased DNA-protein complexes were blocked by excess of unlabeled NF-B oligonucleotide (Fig. 8, lanes 3 and 4) but not by excess of nonspecific unlabeled human distal IL-2 NFAT oligonucleotide (Fig. 8, lanes 5 and 6). To determine the composition of the NF-B complexes, supershift analysis using anti-NF-Bp50, anti-NF-Bp65, or anti-c-Rel Abs was performed. Anti-NF-Bp50 Ab caused the supershift of both bands A and B (Fig. 8, lanes 7 and 8), while anti-NF-Bp65 induced the supershift of only band A (Fig. 8, lanes 9 and 10). Anti-c-Rel Ab failed to react with the DNA-protein complexes (Fig. 8, lanes 11 and 12). These results suggest that band A is composed of p50:p65 heterodimers and band B consists of p50:p50 homodimers. Interestingly, Bryo-1 treatment enhanced the binding of the NF-B subunit p65 while not affecting p50 binding to the NF-B motif (Fig. 8, lanes 2, 6, 10, and 12).

View larger version (54K): [in this window] [in a new window]   FIGURE 8. Gel shift, supershift, and competition analysis of the human IFN-R2 NF-B promoter site. EMSA was performed using a 32P-labeled NF-B oligonucleotide probe in the presence of 7 µg of nuclear extracts prepared from MM6 cells. Before nuclear extract preparation, cells were cultured for 2 h either without stimulation (lanes 1, 3, 5, 7, 9, and 11) or with 1 ng/ml Bryo-1 (lanes 2, 4, 6, 8, 10, and 12). Unlabeled NF-B oligonucleotide was used as a specific competitor (S.C.) with nuclear extracts from medium-treated (lane 3) or Bryo-1-treated MM6 cells (lane 4). Human distal IL-2 NFAT oligonucleotide was used as an unlabeled nonspecific competitor (N.C.) with nuclear extracts from medium-treated (lane 5) or Bryo-1-treated MM6 cells (lane 6). Both the specific and nonspecific competitors were used at 100-fold molar excess. Supershift analysis of NF-B-binding complexes were performed by incubating 7 µg of nuclear extracts from medium-treated or Bryo-1-treated MM6 cells with 200 ng of anti-NF-Bp50 (lanes 7 and 8), anti-NF-Bp65 (lanes 9 and 10), or anti-c-Rel (lanes 11 and 12) Abs. Data shown are from one of two similar experiments.

NF-B inhibitors decrease Bryo-1-induced expression of IFN-R2

To further evaluate the role of NF-B in Bryo-1-induced IFN-R2 mRNA expression, a specific inhibitor of NF-B nuclear translocation, SN50, was used. As depicted in Fig. 9, SN50 inhibits the Bryo-1-enhanced IFN-R2 mRNA expression in monocytes while similar concentration of the inactive peptide SN50M did not block the induction of IFN-R2 mRNA by Bryo-1. These results provide further evidence for a role of NF-B in Bryo-1-enhanced IFN-R2 mRNA expression.

View larger version (14K): [in this window] [in a new window]   FIGURE 9. NF-B inhibitors decrease Bryo-1-induced IFN-R2 mRNA expression. Monocytes were cultured in the presence or absence of 0.01 ng/ml Bryo-1 for 12 h. SN50 or SN50M were added 30 min before Bryo-1 and remained in the culture medium throughout the experiment. Total cellular RNA was isolated, and real-time quantitative RT-PCR for IFN- R2 expression was performed. Column 1: medium-treated cells; column 2: Bryo-1-treated cells; columns 3, 4, and 5: Bryo-1-treated cells and SN50 at 17.5, 35, and 70 µg/ml, respectively; column 6: Bryo-1-treated cells and SN50M at 35 µg/ml. Data shown are from one representative experiment of three performed. The expression of housekeeping gene GAPDH was used as a reference for normalization.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

We demonstrated that the mRNA for IFN-R1 and IFN-R2 was constitutively expressed in MM6 cells, and as little as 1 ng/ml Bryo-1 was sufficient to induce a significant increase in the IFN-R2 mRNA expression (Fig. 1). In contrast, Bryo-1 treatment, at the optimal and achievable dose of 1 ng/ml, did not affect the basal levels of expression of IFN-R1 mRNA in MM6 cells at any of the time points tested (Fig. 2). Even a dose of Bryo-1 as high as 10 ng/ml did not induce IFN-R1 mRNA in MM6 cells (data not shown). These results clearly suggest that the up-regulation of IFN-R2 by Bryo-1 is gene-specific and points toward the IFN-R2 subunit as being the limiting factor in the Bryo-1-induced activation of human monocytic cells in response to IFN-. Our findings support and further extend previous observations by other groups, which have reported that the ability of IFN-R-bearing cells to respond to IFN- can be modulated by regulating the expression of the IFN-R2 subunit (38, 39).

Taken together, these data and our present results suggest that Bryo-1 may play an important role in regulating the ability of monocytic cells to respond to IFN- by specifically enhancing the expression of IFN-R2. However, at the present time, we cannot rule out that postreceptor binding events targeted by Bryo-1 might also contribute to the observed synergistic effects of Bryo-1 and IFN-. The up-regulation of IFN-R2 by Bryo-1 was not restricted to MM6 cells. We demonstrated that Bryo-1 treatment induced an increase in the expression of IFN-R2 mRNA in both the monocytic cell line THP-1 and in primary human monocytes. These results indicate that IFN-R2 gene regulation by Bryo-1 is not an isolated event characteristic of a particular cell line but a phenomenon of broader occurrence.

The Bryo-1-induced up-regulation of IFN-R2 mRNA in MM6 occurred rapidly, being noticeable by 3 h and reaching maximal expression by 6 h post-Bryo-1 treatment. This rapid induction of the IFN-R2 mRNA suggests a direct effect of Bryo-1 on the expression of this gene rather than a secondary effect mediated by a Bryo-1-inducible gene. These results agree with the data from previous reports showing a similar rapid induction of IL-1, IL-6, IL-8, and TNF- mRNA expression in human monocytes stimulated with Bryo-1 (33) and a rapid induction of TNF- mRNA by Bryo-1 in MM6 cells (40). We noticed that THP-1 cells, compared with MM6 cells, required a longer exposure time to respond to Bryo-1. It is possible that the different kinetics observed might be related to different stages of differentiation of these cell lines (27). The Bryo-1-up-regulated IFN-R2 mRNA expression in MM6 cells was dose dependent; as little as 1 ng/ml Bryo-1 was sufficient to significantly enhance the message for IFN-R2. This dose of Bryo-1 is 10- to 1000-fold lower than those used by others to induce activation of monocytic cells (40, 41). Noteworthy, the concentration of Bryo-1 producing an increase in IFN-R2 mRNA is a potentially achievable dose, and it is tempting to speculate that the Bryo-1 up-regulation of IFN-R2 observed in MM6 cells may also be seen in an in vivo setting. Li et al. (41) reported that Bryo-1 was able to up-regulate the level of macrophage CSF receptor in THP-1 cells, and this effect was associated with monocytic differentiation. In agreement with this observation, several studies have previously reported that Bryo-1 promotes the terminal differentiation of both myeloid and lymphocytic cells (42, 43). However, the Bryo-1-augmented expression of IFN-R2 mRNA as we report in the present study is not related to a specific differentiation process because human monocytes are terminally differentiated cells.

Several reports have described the regulation of IFN-R1 gene expression by several stimuli (44). However, little is known about the mechanisms controlling IFN-R2 gene expression. To further dissect the mechanism(s) involved in the Bryo-1-induced IFN-R2 mRNA expression in MM6 cells, run-on transcription assays were performed. Our data showed that the IFN-R2 gene was transcriptionally active in medium-treated cells, and Bryo-1 treatment further enhanced the transcription of this gene by 3-fold. The 5' flanking region of the mouse and human IFN-R2 genes have been cloned and partially characterized, and various putative transcriptional regulatory elements have been identified (32, 34). Since Bryo-1 is known to induce the mRNA of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF- in human monocytes (33) and NF-B have been involved in the transcriptional regulation of these cytokines (45), we performed a nucleotide sequence analysis of the human IFN-R2 promoter (34) using the NF-B sequence found in the mouse IFN-R2 promoter at position –169 to –175 (32). Based on this search, a region (–221 to –230) that matched the previously reported NF-B-like site (GGAAAGTCCC) (46) was identified in the human IFN-R2 promoter. This reversed NF-B-like site is identical to the previously identified sites in the L chain gene intron (–3941 to –3951) (47) and HIV-1 LTR (48). It is noteworthy that the 5' region of the human IFN-R2 gene between the nucleotides –411 to –90, which contains the NF-B site, is important for promoter activity (34).

Our results from EMSA analysis demonstrated that nuclear extracts from medium-treated MM6 cells constitutively expressed NF-B binding activity, which was further enhanced by Bryo-1 treatment. Furthermore, the pattern of NF-B binding suggests that Bryo-1 specifically increased the amount of p65 protein bound to the NF-B motif. In addition, IFN-R2 mRNA expression by Bryo-1-treated human monocytes was significantly decreased in the presence of SN50, a specific inhibitor peptide of NF-B nuclear translocation. In contrast, the inactive control peptide SN50M did not block IFN-R2-enhanced expression by Bryo-1-treated human monocytes. These results support the role of the NF-B motif as a binding site for a nuclear transcription factor enhanced by Bryo-1 treatment in human monocytic cells. Consistent with these results, a recent report by Wang et al. (31) showed that NF-B inhibitors abolished or significantly reduced PMA or Bryo-1-induced TNFR-associated factor 1 expression in human colon cancer cells by PKC activation. Although our results suggest that NF-B plays a role in the Bryo-1-induced IFN-R2 expression in human monocytes, the precise details regarding whether parallel pathways contribute to NF-B activation and, ultimately, IFN-R2 induction are not known. Further work is needed to define the potential effects that Bryo-1 may have on the expression and/or functional status of other regulatory sequences present within the human IFN-R2 promoter such as specificity protein-1 and AP-2 (34).

In an attempt to further assess other mechanisms that might be involved in the accumulation of IFN-R2 mRNA, we investigated the effect of Bryo-1 treatment in IFN-R2 mRNA stability. Bryo-1-treated MM6 cells in the presence of Act D showed a significant increase in the IFN-R2 mRNA t_1/2 as compared with their medium-treated counterparts. Furthermore, IFN-R2 mRNA levels were detectable for up to 8 h after Act D treatment in Bryo-1-treated cells, while the message became almost undetectable by 6 h in medium-treated cells. Similar results were observed in Bryo-1-treated THP-1 cells (data not shown). The increase in the t_1/2 of the IFN-R2 transcripts in Bryo-1-treated cells may account for, or at least in part contribute to, the increased accumulation of IFN-R2 mRNA in MM6 cells. Rapid degradation of RNA encoding many oncogenes and cytokines are in part regulated by A+U-rich element (ARE) sequences in their 3' untranslated region (49, 50). Computer analysis of the published IFN-R2 mRNA sequence (GenBank accession no. NM_005534) (16) revealed one ARE-conserved sequence (AUUUA) located in the 3' untranslated region of the IFN-R2 gene, suggesting that the increased mRNA stability observed in MM6 cells may in part be regulated or controlled by this ARE. Cytoplasmic proteins called adenosine-uridine binding factor have been shown to bind specifically to the AUUUA-conserved sequence and to form exceptionally stable complexes (51). Further work will be needed to determine whether Bryo-1 can induce functionally active adenosine-uridine binding factor. We cannot rule out the possibility that novel regulatory elements or mechanisms other than ARE regions (52) are responsible for or may contribute to the enhanced t_1/2 of the IFN-R2 mRNA observed in MM6 cells. Collectively, these results from the run-on, and mRNA stability experiments indicate that the Bryo-1-induced IFN-R2 mRNA in MM6 cells is regulated by both transcriptional and posttranscriptional mechanisms.

The early up-regulation of IFN-R2 suggested a direct response to Bryo-1, independent of de novo protein synthesis. This interpretation was consistent with the observation that CHX treatment for 6 h did not affect the induction of IFN-R2 mRNA by Bryo-1 in MM6 cells. Interestingly, in the presence of CHX, for 6 h MM6 cells displayed an increase in IFN-R2 mRNA compared with cells grown in the absence of CHX. These results indicate that new protein synthesis is not required for the constitutive or Bryo-1-enhanced expression of IFN-R2 mRNA and also suggest that the basal IFN-R2 expression may be controlled by a de novo synthesized repressor protein(s) and/or by a factor(s) involved in the regulation of mRNA stability. These two mechanisms have been previously suggested to be operating in the superinducibility of cytokine genes by CHX (53, 54, 55).

Several groups have previously reported that phorbol esters are able to induce an increase in IFN-R expression (56). Bryo-1 is a potent ligand and modulator of the phorbol ester receptor PKC, and it can mimic certain effects of the phorbol esters in some biological systems. However, several other properties of Bryo-1 are distinct from the phorbol esters. Of critical importance, unlike the phorbol esters, Bryo-1 lacks tumor-promoting capabilities and actually can counteract tumor promotion induced by phorbol esters (57).

In conclusion, we have demonstrated for the first time that the antineoplastic agent Bryo-1 enhances IFN-R2 mRNA expression in human monocytic cells and in primary human monocytes in a gene-specific manner. We also established that Bryo-1 enhanced IFN-R2 levels through dual mechanisms involving transcriptional and posttranscriptional events that do not require de novo protein synthesis. Finally, we provided evidence suggesting that NF-B might be involved in the transcriptional activation of the IFN-R2 gene.

It has been reported that tumor-bearing hosts have a decreased expression and production of IFN-, which may be partially responsible for the lack of an effective antitumor response (58). It is tempting to speculate that the ability of Bryo-1 to increase IFN-R2 may overcome some of the immune defects observed in cancer patients. These early results warrant further in vivo preclinical research of the combination of Bryo-1 and IFN- in a murine tumor model for potential interventions in cancer immunotherapy.

	Acknowledgments

 
We thank Robert Veith, M.D., Marilyn Schoen, R.N., and Tanya Grersinger, R.N., for their support with the cytapheresis and all the human subjects, for their invaluable contribution and time.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute on Aging, and also by United States Public Health Services Grants R01-AI36450, R01-AI43369, R01-AI59465, and P01-AI57596 from the National Institute of Allergy and Infectious Diseases and Grant RO1-CA46465 from the National Cancer Institute (to S.P.).

² Current address: Lilly Research Laboratories, Eli Lilly, Indianapolis, IN 46285.

³ Address correspondence and reprint requests to Dr. Igor Espinoza-Delgado, Hematology-Oncology Section, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Drive, Room 4C10, Baltimore, MD 21224. E-mail address: espinozaig{at}grc.nia.nih.gov

⁴ Abbreviations used in this paper: Bryo-1, Bryostatin-1; PKC, protein kinase C; IFN-R1, IFN-R1 chain; IFN-R2, IFN-R2 chain; MM6, MonoMac6; Act D, actinomycin D; CHX, cycloheximide; ARE, A+U-rich element.

Received for publication October 21, 2005. Accepted for publication May 27, 2006.

	References

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1. Stone, R. M., E. Sariban, G. R. Pettit, D. W. Kufe. 1988. Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells. Blood 72: 208-213. [Abstract/Free Full Text]
2. Berkow, R. L., A. S. Kraft. 1985. Bryostatin, a non-phorbol macrocyclic lactone, activates intact human polymorphonuclear leukocytes and binds to the phorbol ester receptor. Biochem. Biophys. Res. Commun. 131: 1109-1116. [Medline]
3. Schuchter, L. M., A. H. Esa, S. May, M. K. Laulis, G. R. Pettit, A. D. Hess. 1991. Successful treatment of murine melanoma with bryostatin 1. Cancer Res. 51: 682-687. [Abstract/Free Full Text]
4. Scheid, C., J. Prendiville, G. Jayson, D. Crowther, B. Fox, G. R. Pettit, P. L. Stern. 1994. Immunomodulation in patients receiving intravenous bryostatin 1 in a phase I clinical study: comparison with effects of bryostatin 1 on lymphocyte function in vitro. Cancer Immunol. Immunother. 39: 223-230. [Medline]
5. Drexler, H. G., S. M. Gignac, G. R. Pettit, A. V. Hoffbrand. 1990. Synergistic action of calcium ionophore A23187 and protein kinase C activator bryostatin 1 on human B cell activation and proliferation. Eur. J. Immunol. 20: 119-127. [Medline]
6. Tuttle, T. M., K. P. Bethke, T. H. Inge, C. W. McCrady, G. R. Pettit, H. D. Bear. 1992. Bryostatin 1-activated T cells can traffic and mediate tumor regression. J. Surg. Res. 52: 543-548. [Medline]
7. Curiel, R. E., C. S. Garcia, L. Farouk, M. F. Aguero, I. Espinoza-Delgado. 2001. Bryostatin-1 and IL-2 synergize to induce IFN- expression in human peripheral blood T cells: implications for cancer immunotherapy. J. Immunol. 167: 4828-4837. [Abstract/Free Full Text]
8. Boehm, U., T. Klamp, M. Groot, J. C. Howard. 1997. Cellular responses to interferon . Ann. Rev. Immunol. 15: 749-795. [Medline]
9. Espinoza-Delgado, I., D. L. Longo, G. L. Gusella, L. Varesio. 1992. Regulation of IL-2 receptor subunit genes in human monocytes: differential effects of IL-2 and IFN-. J. Immunol. 149: 2961-2968. [Abstract]
10. Taetle, R., J. M. Honeysett. 1988. -Interferon modulates human monocyte/macrophage transferrin receptor expression. Blood 71: 1590-1595. [Abstract/Free Full Text]
11. Bosco, M. C., I. Espinoza-Delgado, M. Schwabe, G. L. Gusella, D. L. Longo, K. Sugamura, L. Varesio. 1994. Regulation by interleukin-2 (IL-2) and interferon of IL-2 receptor chain gene expression in human monocytes. Blood 83: 2995-3002. [Abstract/Free Full Text]
12. Curiel, R. E., C. S. Garcia, S. Rottschafer, M. C. Bosco, I. Espinoza-Delgado. 1999. Enhanced B7-2 gene expression by interferon in human monocytic cells is controlled through transcriptional and posttranscriptional mechanisms. Blood 94: 1782-1789. [Abstract/Free Full Text]
13. Fleischer, J., E. Soeth, N. Reiling, E. Grage-Griebenow, H. D. Flad, M. Ernst. 1996. Differential expression and function of CD80 (B7-1) and CD86 (B7-2) on human peripheral blood monocytes. Immunology 89: 592-598. [Medline]
14. Pestka, S., S. V. Kotenko, G. Muthukumaran, L. S. Izotova, J. R. Cook, G. Garotta. 1997. The interferon (IFN-) receptor: a paradigm for the multichain cytokine receptor. Cytokine Growth Factor Rev. 8: 189-206. [Medline]
15. Aguet, M., Z. Dembic, G. Merlin. 1988. Molecular cloning and expression of the human interferon receptor. Cell 55: 273-280. [Medline]
16. Soh, J., R. J. Donnelly, S. Kotenko, T. M. Mariano, J. R. Cook, N. Wang, S. Emanuel, B. Schwartz, T. Miki, S. Pestka. 1994. Identification and sequence of an accessory factor required for activation of the human interferon receptor. Cell 76: 793-802. [Medline]
17. Gibbs, V. C., S. R Williams, P. W. Gray, R. D. Schreiber, D. Pennica, G. Rice, D. V. Goeddel. 1991. The extracellular domain of the human interferon receptor interacts with a species-specific signal transducer. Mol. Cell. Biol. 11: 5860-5866. [Abstract/Free Full Text]
18. Hibino, Y., C. S. Kumar, T. M. Mariano, D. H. Lai, S. Pestka. 1992. Chimeric interferon receptors demonstrate that an accessory factor required for activity interacts with the extracellular domain. J. Biol. Chem. 267: 3741-3749. [Abstract/Free Full Text]
19. Rashidbaigi, A., J. A. Langer, V. Jung, C. Jones, H. G. Morse, J. A. Tischfield, J. J. Trill, H. F. Kung, S. Pestka. 1986. The gene for the human immune interferon receptor is located on chromosome 6. Proc. Natl. Acad. Sci. USA 83: 384-388. [Abstract/Free Full Text]
20. Farrar, M. A., J. Fernandez-Luna, R. D. Schreiber. 1991. Identification of two regions within the cytoplasmic domain of the human interferon receptor required for function. J. Biol. Chem. 266: 19626-19635. [Abstract/Free Full Text]
21. Cook, J. R., V. Jung, B. Schwartz, P. Wang, S. Pestka. 1992. Structural analysis of the human interferon receptor: a small segment of the intracellular domain is specifically required for class I major histocompatibility complex antigen induction and antiviral activity. Proc. Natl. Acad. Sci. USA 89: 11317-11321. [Abstract/Free Full Text]
22. Kotenko, S. V., L. S. Izotova, B. P. Pollack, T. M. Mariano, R. J. Donnelly, G. Muthukumaran, J. R. Cook, G. Garotta, O. Silvennoinen, J. N. Ihle. 1995. Interaction between the components of the interferon receptor complex. J. Biol. Chem. 270: 20915-20921. [Abstract/Free Full Text]
23. Soh, J., R. J. Donnelly, T. M. Mariano, J. R. Cook, B. Schwartz, S. Pestka. 1993. Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon receptor: evidence for multiple accessory factors. Proc. Natl. Acad. Sci. USA 90: 8737-8741. [Abstract/Free Full Text]
24. Igarashi, K., G. Garotta, L. Ozmen, A. Ziemiecki, A. F. Wilks, A. G. Harpur, A. C. Larner, D. S. Finbloom. 1994. Interferon induces tyrosine phosphorylation of interferon receptor and regulated association of protein tyrosine kinases: Jak1 and Jak2, with its receptor. J. Biol. Chem. 269: 14333-14336. [Abstract/Free Full Text]
25. Krause, C. D., E. Mei, J. Xie, Y. Jia, M. A. Bopp, R. M. Hochstrasser, S. Pestka. 2002. Seeing the light: preassembly and ligand-induced changes of the interferon receptor complex in cells. Mol. Cell. Proteomics 1: 805-815. [Abstract/Free Full Text]
26. Taylor, L. S., G. W. Cox, G. Melillo, M. C. Bosco, I. Espinoza-Delgado. 1997. Bryostatin-1 and IFN- synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages. Cancer Res. 57: 2468-2473. [Abstract/Free Full Text]
27. Ziegler-Heitbrock, H. W., E. Thiel, A. Futterer, V. Herzog, A. Wirtz, G. Riethmuller. 1988. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int. J. Cancer 41: 456-461. [Medline]
28. Beg, A. A., D. Baltimore. 1996. An essential role for NF-B in preventing TNF- induced cell death. Science 274: 782-784. [Abstract/Free Full Text]
29. Zhang, Y., J. X. Lin, J. Vilicek. 1990. Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a b-like sequence. Mol. Cell. Biol. 10: 3818-3823. [Abstract/Free Full Text]
30. Orlowski, R. Z., A. S. Baldwin, Jr. 2002. NF-B as a therapeutic target in cancer. Trends Mol. Med. 8: 385-389. [Medline]
31. Wang, X., Q. Wang, W. Hu, B. M. Evers. 2004. Regulation of phorbol ester-mediated TRAF1 induction in human colon cancer cells through a PKC/RAF/ERK/NF-B dependent pathway. Oncogene 23: 1885-1895. [Medline]
32. Ebensperger, C., S. Rhee, G. Muthukumaran, D. Lembo, R. Donnelly, S. Pestka, Z. Dembic. 1996. Genomic organization and promoter analysis of the gene ifngr2 encoding the second chain of the mouse interferon receptor. Scand. J. Immunol. 44: 599-606. [Medline]
33. Bosco, M. C., S. Rottschafer, L. S. Taylor, J. R. Ortaldo, D. L. Longo, I. Espinoza-Delgado. 1997. The antineoplastic agent bryostatin-1 induces proinflammatory cytokine production in human monocytes: synergy with interleukin-2 and modulation of interleukin-2R chain expression. Blood 89: 3402-3411. [Abstract/Free Full Text]
34. Rhee, S., C. Ebensperger, Z. Dembic, S. Pestka. 1996. The structure of the gene for the second chain of the human interferon receptor. J. Biol. Chem. 271: 28947-28952. [Abstract/Free Full Text]
35. Cippitelli, M., A. Sica, V. Viggiano, J. Ye, P. Ghosh, M. J. Birrer, H. A. Young. 1995. Negative transcriptional regulation of the interferon promoter by glucocorticoids and dominant negative mutants of c-Jun. J. Biol. Chem. 270: 12548-12556. [Abstract/Free Full Text]
36. Hamalainen, H., S. Meissner, R. Lahesmaa. 2000. Signaling lymphocytic activation molecule (SLAM) is differentially expressed in human Th1 and Th2 cells. J. Immunol. Methods 242: 9-19. [Medline]
37. Grant, S., J. Roberts, E. Poplin, M. B. Tombes, B. Kyle, D. Welch, M. Carr, H. D. Bear. 1998. Phase Ib trial of bryostatin 1 in patients with refractory malignancies. Clin. Cancer Res. 4: 611-618. [Abstract]
38. Pernis, A., S. Gupta, K. J. Gollob, E. Garfein, R. L. Coffman, C. Schindler, P. Rothman. 1995. Lack of interferon receptor chain and the prevention of interferon signaling in TH1 cells. Science 269: 245-247. [Abstract/Free Full Text]
39. Novelli, F., P. Bernabei, L. Ozmen, L. Rigamonti, A. Allione, S. Pestka, G. Garotta, G. Forni. 1996. Switching on of the proliferation or apoptosis of activated human T lymphocytes by IFN- is correlated with the differential expression of the - and -chains of its receptor. J. Immunol. 157: 1935-1943. [Abstract]
40. Steube, K. G., H. G. Drexler. 1995. The protein kinase C activator bryostatin-1 induces the rapid release of TNF- from MONO-MAC-6 cells. Biochem. Biophys. Res. Commun. 214: 1197-1203. [Medline]
41. Li, Y., R. M. Mohammad, A. al Katib, M. L. Varterasian, B. Chen. 1997. Bryostatin 1 (bryo1)-induced monocytic differentiation in THP-1 human leukemia cells is associated with enhanced c-fyn tyrosine kinase and M-CSF receptors. Leuk. Res. 21: 391-397. [Medline]
42. Lilly, M., C. Brown, G. Pettit, A. Kraft. 1991. Bryostatin 1: a potential anti-leukemic agent for chronic myelomonocytic leukemia. Leukemia 5: 283-287. [Medline]
43. Mohammad, R. M., H. Diwakaran, A. Maki, M. A. Emara, G. R. Pettit, B. Redman, A. al Katib. 1995. Bryostatin 1 induces apoptosis and augments inhibitory effects of vincristine in human diffuse large cell lymphoma. Leuk. Res. 19: 667-673. [Medline]
44. Fischer, T., K. Wiegmann, H. Bottinger, K. Morens, G. Burmester, K. Pfizenmaier. 1990. Regulation of IFN- receptor expression in human monocytes by granulocyte-macrophage colony-stimulating factor. J. Immunol. 145: 2914-2919. [Abstract]
45. Mukaida, N., Y. Mahe, K. Matsushima. 1990. Cooperative interaction of nuclear factor B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J. Biol. Chem. 265: 21128-21133. [Abstract/Free Full Text]
46. Karin, M., Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-B activity. Annu. Rev. Immunol. 18: 621-663. [Medline]
47. Max, E. E., J. V. Maizel, Jr, P. Leder. 1981. The nucleotide sequence of a 5.5-kilobase DNA segment containing the mouse immunoglobulin J and C region genes. J. Biol. Chem. 256: 5116-5120. [Abstract/Free Full Text]
48. Sica, A., T. H. Tan, N. Rice, M. Kretzschmar, P. Ghosh, H. A. Young. 1992. The c-rel protooncogene product c-Rel but not NF-B binds to the intronic region of the human interferon gene at a site related to an interferon-stimulable response element. Proc. Natl. Acad. Sci. USA 89: 1740-1744. [Abstract/Free Full Text]
49. DeMaria, C. T., G. Brewer. 1996. AUF1 binding affinity to A+U-rich elements correlates with rapid mRNA degradation. J. Biol. Chem. 271: 12179-12184. [Abstract/Free Full Text]
50. Shaw, G., R. Kamen. 1986. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46: 659-667. [Medline]
51. Guhaniyogi, J., G. Brewer. 2001. Regulation of mRNA stability in mammalian cells. Gene 265: 11-23. [Medline]
52. Hel, Z., E. Skamene, D. Radzioch. 1996. Two distinct regions in the 3' untranslated region of tumor necrosis factor mRNA form complexes with macrophage proteins. Mol. Cell. Biol. 16: 5579-5590. [Abstract]
53. Sariban, E., K. Imamura, R. Luebbers, D. Kufe. 1988. Transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes. J. Clin. Invest. 81: 1506-1510. [Medline]
54. Fenton, M. J., M. W. Vermeulen, B. D. Clark, A. C. Webb, P. E. Auron. 1988. Human pro-IL-1 gene expression in monocytic cells is regulated by two distinct pathways. J. Immunol. 140: 2267-2273. [Abstract]
55. Whittemore, L. A., T. Maniatis. 1990. Postinduction repression of the interferon gene is mediated through two positive regulatory domains. Proc. Nat. Acad. Sci. USA 87: 7799-7803. [Abstract/Free Full Text]
56. Sakamoto, S., J. Nie, T. Taniguchi. 1999. Cutting edge: phorbol ester induction of IFN- receptors leads to enhanced DR gene expression. J. Immunol. 162: 4381-4384. [Abstract/Free Full Text]
57. Hennings, H., P. M. Blumberg, G. R. Pettit, C. L. Herald, R. Shores, S. H. Yuspa. 1987. Bryostatin 1, an activator of protein kinase C, inhibits tumor promotion by phorbol esters in SENCAR mouse skin. Carcinogenesis 8: 1343-1346. [Abstract/Free Full Text]
58. Ghosh, P., A. Sica, H. A. Young, J. Ye, J. L. Franco, R. H. Wiltrout, D. L. Longo, N. R. Rice, K. L. Komschlies. 1994. Alterations in NF-B/Rel family proteins in splenic T cells from tumor-bearing mice and reversal following therapy. Cancer Res. 54: 2969-2972. [Abstract/Free Full Text]

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