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[Cancer Research 65, 7403-7412, August 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Signaling through IFN Regulatory Factor-5 Sensitizes p53-Deficient Tumors to DNA Damage–Induced Apoptosis and Cell Death

Guodong Hu, Margo E. Mancl and Betsy J. Barnes

Division of Viral Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland

Requests for reprints: Betsy J. Barnes, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231. Phone: 443-287-2758; Fax: 410-955-0840; E-mail: barnebe{at}jhmi.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human IFN regulatory factor-5 (IRF-5) is a candidate tumor suppressor gene that mediates cell arrest, apoptosis, and immune activation. Here we show that ectopic IRF-5 sensitizes p53-proficient and p53-deficient colon cancer cells to DNA damage–induced apoptosis. The combination IFN-ß and irinotecan (CPT-11) cooperatively inhibits cell growth and IRF-5 synergizes with it to further promote apoptosis. The synergism is due to IRF-5 signaling since a striking defect in apoptosis and cell death was observed in IRF-5-deficient cells, which correlated well with a reduction in DNA damage–induced cellular events. Components of this IRF-5 signaling pathway are investigated including a mechanism for DNA damage–induced IRF-5 activation. Thus, IRF-5–regulated pathways may serve as a target for cancer therapeutics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Swift elimination of undesirable cells is an important feature in tumor suppression and immunity. The tumor suppressor p53 and IFN-{alpha}, -ß, and -{gamma} are essential for the induction of apoptosis in cancer cells and in antiviral immune responses, respectively, but little is known about their interrelationship. It has recently been suggested that there is cross-talk between the p53 and IFN signaling pathways (1), but the transcriptional targets that may be responsible for the integration of these two pathways are currently unknown. Recent data from other laboratories and ours indicate that members of the IFN regulatory factor (IRF) family, specifically IRF-1 and IRF-5, have essential roles in both p53- and IFN-mediated cell growth regulation and cell death. IRF-1 and IRF-5 are both targets of IFN transcriptional regulation (2, 3). However, while IRF-1 and p53 are coordinately up-regulated during the response to DNA damage and cooperate in the regulation of the cyclin-dependent kinase inhibitor p21 (WAF1/CIP1), IRF-5 is a direct target of p53 (4), yet its cell cycle regulatory and proapoptotic effects are p53 independent (3). IRF-1 has also been shown to play an essential role in mediating apoptosis and tumor suppressor activity in the absence of p53 (5–7).

Although originally identified as regulators of IFN and IFN-stimulated genes, IRFs have also been shown to be involved in the regulation of natural immunity against viruses, acquired immunity, cell differentiation, apoptosis, embryogenesis, growth regulation, and tumorigenesis (8). Currently, nine IRF family members have been described in mammals: IRF-1 through IRF-7, IRF-8, also called IFN consensus sequence-binding protein, and IRF-9, originally described as IFN-stimulated gene factor 3 {gamma}. Although the role of IRFs in response to viral infection is well established, the precise mechanism(s) underlying IRF-mediated tumor suppression and/or oncogenesis remain to be elucidated. With regard to tumor suppressor activity, IRF-1, in particular, has received much attention over the past 10 years. Regulation of IRF-1 is thought to be controlled by two distinct signaling pathways; a Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway in virus-infected or IFN-treated cells and an ataxia telangiectasia mutated (ATM) signaling pathway in DNA-damaged cells (9). Genes downstream of IRF-1 in these two pathways include IFNB (10, 11), RNA-dependent protein kinase (12, 13), 2-5A synthetase (14, 15), cyclin-dependent kinase inhibitor p21 (WAF1/CIP1; refs. 16, 17), caspase-1 and caspase-3 (18), and Apo2L/TRAIL (19). IRF-1 has been shown to mediate retinoid and IFN antitumor signaling to death ligand Apo2L/tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) through binding to functional IRF binding elements and IFN-stimulated response elements in the Apo2L/TRAIL promoter (20). Recent data also implicate IRF-1 as a critical transducer of IFN-{gamma}–mediated Apo2L/TRAIL-induced apoptosis (19) and tamoxifen-mediated apoptosis (18).

Although the role of IRF-5 in the antiviral immune response is well established (21–27), little is known about its response to other environmental stresses, such as DNA damage. There are several lines of evidence that indicate IRF-5 is a direct target of p53 (3, 4). However, overexpression of IRF-5 or up-regulation by type I IFNs induced a G2-M cell arrest and cell death in B-cell lymphomas expressing nonfunctional p53 (3). Several cell cycle regulatory and proapoptotic genes were shown to be directly or indirectly modulated by IRF-5, including p21, caspase-8, Bak, Bax, and DAP kinase 2 (3). Furthermore, like IRF-1, deletion and alternative splicing of IRF-5 pre-mRNA have been observed in multiple primary and immortalized hematologic malignancies, as well as in solid phase tumors of the gastrointestinal tract1 (3, 27), providing evidence for tumor suppressor activities.

The current study investigates potential mechanisms by which IRF-5 is a critical mediator of DNA damage– and IFN-induced signaling. The DNA damaging agent irinotecan (CPT-11) and type I IFNs cooperate in the transcriptional regulation of IRF-5. Evidence is presented that IRF-5 is required for CPT-11–and IFN plus CPT-11 combination–mediated cell growth inhibition and apoptosis, which is p53 independent. Overexpression of IRF-5 in p53-deficient tumor cells sensitized them to DNA damage–mediated apoptosis. Here we show by immunoblot and microarray analysis in conjunction with targeted short interfering RNAs (siRNA), that IRF-5 is critical for the induction/activation of a specific profile of CPT-11–regulated genes. In addition, the mechanism(s) of DNA damage–induced IRF-5 activation is discussed. Our results suggest that targeting/activating the IRF-5 signaling pathway or components of this pathway, will provide a synergistic induction of tumor cell death in combination with conventional chemotherapeutic agents. This work also supports the possible clinical utility of IFN combination therapies for cancer treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and transfection. The HCT116 colon adenocarcinoma cell line and the isogenic p53-deficient (p53–/–) derivative of HCT116 (28) were used. HCT116 p53–/– cells were transfected with an expression vector encoding myc-IRF-5 V3/4 (pcDNA3.1), gfp-IRF-1, or gfp-IRF-5 V3/4 (21, 27), or with an empty gfp control vector using FuGene 6 (Roche, Indianapolis, IN) and cells overexpressing each gfp fusion protein were isolated by selection of gfp-positive cells using fluorescence-activated cell sorting. Human HCT116 and DLD1 colon carcinoma cells were cultured at 37°C and 5% CO2 in McCoy's medium supplemented with 10% FCS, penicillin, and streptomycin (100 units/mL); stable gfp-expressing cell lines were maintained in 1 mg/mL G418-McCoy's selection medium; human A549 lung carcinoma cells were grown in DMEM supplemented with 10% FCS.

Treatment with irinotecan (CPT-11), type I IFNs, or Newcastle disease virus. Cells were incubated with irinotecan hydrochloride (50 µg/mL; Camptosar, Pharmacia-Upjohn, Kalamazoo, MI) or type I IFNs (IFN-{alpha}, -ß, or -{gamma}; PBL Biomedical Laboratories, Piscataway, NJ) at 1,000 units/mL at 37°C for the indicated time intervals. HCT116 cells were infected with Newcastle disease virus (ATCC no. VR-699) at 100 hemagglutinin units/mL.

Immunoprecipitation and immunoblot analysis. Cell lysates for immunoprecipitation were prepared as described (23); 300 µg extracts were incubated with 1 to 2 µg of the indicated antibodies for 16 hours at 4°C [phospho-threonine, phospho-tyrosine (Cell Signaling, Beverly, MA); phospho-serine (BioSource, Camarillo, CA); phospho-IRF-5 (Ser-427/Ser-430; Affinity Biosciences, Golden, CO)], bound to protein G-Sepharose beads for 2 hours at 4°C. Precipitates were washed thrice and eluted by boiling in 1x SDS loading buffer. Cell lysates were prepared as described (24); 50 µg of whole cell lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and probed with antibodies against IRF-1 (BD Biosciences, San Jose, CA), IRF-5 (Abcam, Cambridge, MA), Bak, Bax, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, cleaved caspase-8, cleaved poly(ADP-ribose) polymerase (PARP), and actin (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive protein complexes were visualized with enhanced chemiluminescence by ECL reagents (Amersham Biosciences, Piscataway, NJ). Membranes were stripped and reprobed multiple times using Restore Western Blot stripping buffer (Pierce, Rockford, IL).

RNA interference. The coding region of IRF-1 or IRF-5 was targeted with the following siRNA sequences: IRF-1, 5'-GACGTGGAAGGCCAACTTT-3'; IRF-5, 5'-ATACACCGAAGGCGTGGAT-3' (Dharmacon, Inc., Lafayette, CO). The efficiency of IRF-1 or IRF-5 gene silencing was established in HCT116 cells (six-well plates, 40% confluency) as described (25) using Mirus TransIT TKO reagent (Mirus, Madison, WI). Transfection efficiencies were determined by fluorescent microscropy to be ~80% after cotransfection with IRF-5 siRNA and a gfp control plasmid. Cells were harvested 24 hours following transfection for reverse transcription-PCR (RT-PCR) and immunoblot analysis (3). The effect of IRF-1 or IRF-5 gene silencing was analyzed by transfection of HCT116 cells with 10 or 5 nmol/L of IRF-1 or IRF-5 siRNA, respectively. Eight hours following transfection, cells were treated with CPT-11 for an additional 16 hours and then harvested for extraction of RNA and protein. Total RNA was analyzed by RT-PCR (24), by RNase protection assay using hAPO-1c and hAPO-2b multiprobe template sets (BD PharMingen, Lexington, KY; refs. 3, 22), or by microarray using the Cancer Pathway Finder GEArray Q series microarrays (SuperArray, Inc., Bethesda, MD; ref. 3). The effect of IRF-1 or IRF-5 siRNA on protein expression was analyzed by immunoblot.

Analysis of cell death and apoptosis. Cells were assessed for morphologic features of apoptosis (condensed chromatin and micronucleation) by microscopic visualization. Cell viability was determined by analysis of propidium iodide (BD PharMingen) staining of harvested cells (adherent + floating in the medium) using a flow cytometer (Becton Dickinson, Palo Alto, CA). The average percent viability (mean ± SE) was calculated from three different experiments. Apoptosis was quantified by single Annexin V-FITC or Annexin V-phycoerythrin staining (BD PharMingen) on a flow cytometer.

Subcellular localization of gfp-IRF-5 proteins. HCT116 p53–/– cells stably overexpressing gfp-IRF-5 or p53+/+ cells transiently transfected with gfp-IRF-5 expression plasmid using FuGene 6 transfection reagent (Roche) were visually analyzed for IRF-5 nuclear translocation. After 30 hours, cells were split and left unstimulated or stimulated with CPT-11 for the indicated time intervals. After stimulation, cells were fixed with 4% paraformaldehyde, mounted with ProLong antifade reagent (Molecular Probes, Inc., Eugene, OR), stained with 4',6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA), and examined by fluorescent microscopy using a Nikon TE-200 and DXM12000F at x40 magnification.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN regulatory factor-5 sensitizes p53-deficient HCT116 tumor cells to DNA damage–mediated apoptosis. As a single agent, the topoisomerase I inhibitor irinotecan (CPT-11, Camptosar) is most effective in suppressing cell growth or inducing apoptosis in p53-proficient tumors (Fig. 1A-C). CPT-11 is currently used in combination with 5-fluorouracil and leucovorin for postoperative treatment or in current or advanced colorectal cancer (29, 30). DNA damage–induced signaling by CPT-11 is thought to be dependent on p53, as are many chemotherapeutic agents, and thus are not effective as single agents in p53-deficient tumors (31). We have examined a new combinatorial therapeutic approach, IFN-ß and CPT-11 treatment, which revealed a significant increase in the number of p53+/+ (data not shown) and p53–/– cells undergoing apoptosis, through Annexin V-phycoerythrin staining, compared with the single CPT-11 treatment (Fig. 1C and D). p53-deficient cells exposed to IFN-ß and CPT-11 for 16 hours increased the number of cells undergoing apoptosis from 10% to 32%.



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  		Figure 1. Overexpression of IRF-5 enhances CPT-11–mediated apoptosis in p53-deficient cells. A, untreated HCT116 cells were stained with Annexin V-phycoerythrin and analyzed by flow cytometry. B, p53-proficient HCT116 cells are sensitive to DNA damage–mediated apoptosis by CPT-11 (16 hours). C, p53-deficient cells are relatively insensitive to DNA damage–mediated apoptosis by CPT-11. D, the number of cells undergoing apoptosis is enhanced by combination IFN-ß and CPT-11 in p53–/– cells. E, Annexin V-phycoerythrin staining of untreated p53–/– cells stably expressing gfp-IRF-5. Bottom right, population of gfp-IRF-5–positive cells. F, IRF-5 sensitizes HCT116 p53–/– tumor cells to DNA damage–mediated apoptosis. G, apoptosis is further enhanced by IFN-ß plus CPT-11 combination treatment in gfp-IRF-5–expressing p53–/– cells. Top, percentage of cells stained positive for phycoerythrin.

 
It was previously shown that generation of a stable IRF-5–overexpressing B-cell lymphoma cell line with nonfunctional p53 induced spontaneous apoptosis (3). To explore the role of IRF-5 in DNA damage– and/or IFN-mediated apoptosis, we generated p53-deficient HCT116 cells that stably overexpress gfp-IRF-5. In the RNase protection assay, p21, Bax, Bak, and caspase-8 gene expression was up-regulated in these cells compared with gfp-expressing vector control cells (data not shown). When stained with Annexin V-phycoerythrin, stable gfp-IRF-5–expressing cells were identical to gfp vector control cells in that neither cell line was undergoing substantial amounts of apoptosis (Fig. 1E and data not shown). However, overexpression of gfp-IRF-5 in p53-deficient cancer cells sensitized them to CPT-11–induced apoptosis (Fig. 1E and F), similar to the levels observed in IFN-ß– and CPT-11–treated cells (Fig. 1D). When the gfp-IRF-5–overexpressing cells were treated with IFN-ß and CPT-11, the number of cells undergoing apoptosis was further elevated (Fig. 1G), indicating that combinatorial therapies targeting IRF-5 transcriptional activity or signaling may enhance the sensitivity of tumor cells to undergo apoptosis and cell death. The levels of apoptosis induced by CPT-11 in p53-proficient (38%) and p53-deficient cells expressing gfp-IRF-5 (30%) were nearly identical, suggesting that IRF-5 can compensate for p53 in these cells, thus resensitizing both p53-proficient and p53-deficient cells to DNA damage–mediated apoptosis.

IFN regulatory factor-5 is a critical transducer of DNA damage–mediated cell death and apoptosis. To evaluate the function and/or contribution of IRF-5 (and IRF-1) signaling to type I IFN– or DNA damage–mediated apoptosis, we have generated targeted siRNAs that are specific to either. To date, the majority of functions characterized for human IRF-5 have been determined by overexpression assays (3, 4, 21–24). Figure 2A shows that the targeted elimination of endogenous IRF-5 or IRF-1 mRNA with siRNAs is specific and does not affect ISG20 gene expression (32). A similar effect was observed in both HCT116 cell lines: 10 nmol/L IRF-1 siRNA was optimal for >90% knockdown effect, 5 nmol/L IRF-5 siRNA was sufficient to knockdown >90% expression in p53-proficient cells, and 7 nmol/L was necessary in p53-deficient cells (data not shown).



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  		Figure 2. IRF-5 is a critical transducer of DNA damage–mediated cell death and apoptosis. A, generation of IRF-1- and IRF-5-specific "knockdowns" using targeted siRNAs. HCT116 cells were transfected with either 5 nmol/L IRF-5 siRNA (left) or 10 nmol/L IRF-1 siRNA (right), as indicated, and then left untreated or treated with CPT-11 for 16 hours. RNA was isolated and RT-PCR done. B, IRF-5 siRNA reduces the levels of DNA damage–mediated cell death, as determined by propidium iodide staining. Cells were treated with CPT-11 for 24 hours or with CPT-11 and IFN-ß in the presence or absence of IRF-5 siRNA. Right, histogram of data. C, transfection of HCT116 cells with IRF-5 siRNA gives a dramatic decrease in CPT-11–mediated apoptosis compared with IRF-1 siRNA transfection, as determined by Annexin V-FITC staining. The double IRF-1 and IRF-5 knockdown abrogated CPT-11–induced apoptosis.

 
Overexpression of IRF-5 in a B-cell lymphoma has been shown to inhibit colony formation on soft agar (3, 4) and induce apoptosis (3). Here, we instead examined the effect of knocking down endogenous IRF-5 expression on CPT-11–induced cell death. Treatment of p53+/+ cells with CPT-11 for 24 hours caused 65% of cells to undergo cell death as determined by propidium iodide staining, whereas transfection with IRF-5 siRNA reduced the levels of CPT-11–induced cell death to 37% (Fig. 2B). There was no difference in the percentage of cells undergoing cell death in the untreated or untransfected and IRF-5 siRNA–transfected cells. The percentage of cells undergoing CPT-11–induced cell death was substantially elevated when treated with the combination of IFN-ß and CPT-11 for 24 hours (>80%). Treatment with IFN-ß alone gave ~15% to 20% cell death (data not shown). Transfection with IRF-5 siRNA protected cells from IFN-ß and CPT-11 combination–induced cell death (~28%; Fig. 2B). These data indicate that IRF-5 is a critical transducer of DNA damage–induced cell death and also contributes to the observed combinatorial cytotoxic effects of IFN-ß and CPT-11.

We next examined the effects of IRF-1 and IRF-5 siRNA on CPT-11–mediated apoptosis by Annexin V-FITC staining. p53+/+ cells were either left untransfected or transfected with a nonspecific LacZ siRNA, IRF-1 siRNA, or IRF-5 siRNA, and then treated with CPT-11 for 24 hours or left untreated. The percent of Annexin V-FITC–positive cells was determined by flow cytometry, as shown in Fig. 2C. Approximately 85% of untransfected or LacZ transfected cells underwent apoptosis when treated with CPT-11. Transfection of IRF-1 siRNA gave a reduction of ~15% whereas IRF-5 siRNA reduced the levels of CPT-11–induced apoptosis by nearly 50%. The combined effect of IRF-1 and IRF-5 siRNAs further reduced the levels of Annexin V-FITC–stained cells to that observed in untreated HCT116 (Fig. 2C). Similar effects were observed in human A549 lung carcinoma and DLD1 colon carcinoma cell lines that express endogenous IRF-5 (data not shown).

p53-independent induction of IFN regulatory factor-1 and IFN regulatory factor-5 by IFN or CPT-11. We and others have recently shown that IRF-5 is a downstream component of the p53 signaling cascade (3, 4). In addition, IRF-5 is an IFN-inducible transcription factor that also mediates virus-induced type I IFN gene expression (3, 24, 27). When overexpressed in a B-cell lymphoma, IRF-5 induced a G2-M cell cycle arrest and apoptosis that was p53 independent; overexpression also inhibited both in vitro and in vivo tumor cell growth (3) and HCT116 or A549 colony formation on soft agar (4). Here, we begin to elucidate the function of endogenous IRF-5 in DNA damage signaling. IRF-5 gene expression was examined in HCT116 colon cancer cells treated over a time course with CPT-11. Expression was up-regulated as early as 8 hours posttreatment and levels were further enhanced by 16 hours (Fig. 3A). Similar findings were observed in the p53–/– isogenic derivatives (data not shown). Treatment of HCT116 cells with type I ({alpha} and ß) or type II ({gamma}) IFN for 8 hours induced IRF-1, IRF-5, and p21 transcript levels in a manner expected in cells with intact IFN signaling components (i.e., JAK/STATs). IFN-{gamma} was a more efficient inducer of IRF-1 gene expression whereas IFN-{alpha} (data not shown) or IFN-ß was a more effective inducer of IRF-5, and p21 was equally well induced by IFN-ß or IFN-{gamma} (Fig. 3B). To determine whether the observed induction was dependent on p53, IRF-1 and IRF-5 gene expression was examined in both p53-proficient and p53-deficient derivatives treated with CPT-11. Results indicate that transcriptional regulation of these two IRF family members by DNA damaging agents is p53 independent (Fig. 3B; refs. 3, 9). In addition, p21 transcripts were detected in both p53-proficient and p53-deficient cells treated with CPT-11, yet levels were significantly reduced in p53-deficient cells (Fig. 3B).



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  		Figure 3. IRF-5 transcripts are up-regulated by the topoisomerase I inhibitor CPT-11. A, p53-proficient HCT116 cells were left untreated or treated with CPT-11 for the indicated time intervals. Transcript levels were determined by RT-PCR. B, induction of endogenous IRF-1, IRF-5, and p21 transcripts by IFNs or CPT-11 is p53 independent. HCT116 isogenic cells were left untreated (con) or treated with IFN-ß, IFN-{gamma}, or CPT-11 for 16 hours. RNA was isolated and RT-PCR done. C, IRF-5 transcripts were further enhanced in p53-deficient cells by IFN-ß and CPT-11 combination treatment. HCT116 p53–/– cells were treated for 16 hours as indicated; either left unstimulated (con), treated with IFN-ß or CPT-11, or incubated with both IFN-ß and CPT-11. RNA was isolated and RT-PCR done for IRF-1, IRF-5, and ß-actin.

 
The transcription factor IRF-1 is a regulator of IFN-{gamma} and has recently been shown to mediate IFN-{gamma}–induced apoptosis (19). Because studies from a number of laboratories have shown an important role for IRF-1 in IFN-{gamma}– or DNA damage–mediated apoptosis, we have focused instead on evaluating IRF-5 as a potential new mediator of type I IFN and DNA damage signaling that leads to apoptosis and/or cell death. As shown in Fig. 3C, IRF-5 is specifically up-regulated by IFN-ß and levels were dramatically increased by the IFN-ß and CPT-11 combination in p53-deficient cells. A similar effect was observed with IFN-{alpha} and CPT-11 in p53+/+ cells (data not shown).

Mechanism of IFN regulatory factor-5–mediated DNA damage signaling. We next examined the mechanism of IRF-5–mediated sensitization of p53+/+ and p53–/– cells to DNA damage–induced apoptosis. First, the kinetics of CPT-11–induced apoptosis was analyzed by immunoblot. Exposure of p53+/+ cells to CPT-11 for the indicated times resulted in elevated Bax levels and activation of caspase-3, caspase-7, and PARP by substrate cleavage at 16 hours and protein levels were further enhanced at 24 hours posttreatment (Fig. 4A). Low levels of caspase-3 and caspase-7 cleavage were observed as early as 8 hours post CPT-11 treatment. This induction correlated well with the induction of IRF-5 transcripts by CPT-11 at 8 hours (Fig. 3A). We next examined the effect of IRF-5 siRNA on proapoptotic protein expression/activation by CPT-11. In untransfected cells treated with CPT-11 for 16 hours, endogenous IRF-5 protein levels and PARP, caspase-3, caspase-7, and caspase-8 cleavage were induced (Fig. 4B). Transfection of HCT116 with IRF-5 siRNA had no effect on protein expression in untreated cells, except for constitutively expressed Bax (lanes 1 and 3), which was reduced in both untreated and CPT-11–treated cells (lanes 1 and 2). Moreover, IRF-5 siRNA dramatically reduced the levels of CPT-11–induced caspase-3, caspase-8, and PARP cleavage. Interestingly, whereas caspase-7 cleavage was induced by CPT-11, the levels were unaffected by transfection with IRF-5 siRNA, suggesting that signaling via IRF-5 goes through caspase-3 and not caspase-7. Furthermore, data indicate that IRF-5 is upstream of Bax and caspase-8 apoptotic signaling.



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  		Figure 4. Mechanistic role for IRF-5 in DNA damage–mediated apoptosis. A, HCT116 p53+/+ cells were treated with CPT-11 for the indicated time intervals. Immunoblot analysis of whole cell lysates was done with the indicated antibodies. A single blot was stripped and reprobed multiple times. Representative of at least two independent experiments. B, knockdown of IRF-5 protein expression with targeted siRNA dramatically inhibits CPT-11–mediated caspase and PARP cleavage. HCT116 p53+/+ cells were transfected with IRF-5 siRNA and left untreated (5siR) or treated with CPT-11 (5siR CPT) for 16 hours, or transfected with LacZ siRNA (con) and left untreated or treated with CPT-11 (CPT), as indicated. Proteins were detected with antibodies that recognize the cleaved active forms of caspases or PARP. Representative of three independent experiments. C, CPT-11–mediated induction of proapoptotic genes Bak, Bax, caspase-3, and caspase-7 occurs primarily through the IRF-5 signaling pathway. Analysis of the effect of CPT-11, IRF-5, or IRF-1 siRNA on proapoptotic gene expression by the RNase protection assay. Representative of three independent experiments; same exposure time. D, immunoblot analysis of the effect of IRF-1 siRNA on Bak, Bax, caspase-3, and caspase-7 protein expression.

 
The effect of CPT-11 on the expression of IFN-responsive proapoptotic genes in p53+/+ cells was examined to determine whether transcriptional regulation occurred via IRF-5 signaling (Fig. 4C). CPT-11–induced expression of Bak, Bax, caspase-3, and caspase-7 was impaired by targeted elimination of IRF-5 siRNA (Fig. 4C, left). IRF-1 siRNA gave only a reduction of caspase-3 mRNA levels in CPT-11–treated cells, whereas in untreated cells, the targeted siRNA reduced levels of Bak and Bax (Fig. 4C, right). To confirm these findings, we examined protein levels in untreated and treated cells. Figure 4D shows that IRF-1 siRNA had a generally low (Bak and caspase-3) to undetectable effect (Bax and caspase-7) on proapoptotic protein expression. Whereas the IRF-5 siRNA had no effect on caspase-7 protein levels (data not shown) or the levels of CPT-11–induced cleavage (Fig. 4B), IRF-5 may be involved in the transcriptional regulation of this gene because mRNA levels were dramatically reduced by IRF-5 siRNA. Together, these data indicate that IRF-5, and not IRF-1, is the primary mediator of CPT-11–induced DNA damage signaling.

Gene expression was further analyzed by microarray using the Human Cancer Pathway Finder array. Total RNA (5 µg) was isolated from p53+/+ cells in the presence or absence of transfected IRF-5 siRNA in conjunction with CPT-11 treatment for 16 hours. cDNA was transcribed and 32P-labeled probes were generated for hybridization to each array. A subtraction analysis was done to determine CPT-11–induced genes that were dependent on IRF-5 signaling. This particular array was used because it contains some of the previously identified and confirmed p53 and IRF-5 (3, 24) target genes (i.e., p21, p53, mdm-2, and IFNB, caspase-8, Bax, respectively), which served as positive controls for either gene. Table 1 gives a list of the genes that were modulated by CPT-11 independent of IRF-5 expression, and those in which expression was dramatically affected by a loss of IRF-5 signaling, thus indicating a critical role for IRF-5 in the expression of CPT-11–modulated genes. Genes of which expression seemed to be directly or indirectly dependent on IRF-5 expression were genes involved in cell cycle control and DNA damage repair (BRCA1, cyclin D1, cdc25A, CDK4, p27, DNA-PK, and chk2), apoptosis and cell senescence (Apaf1, Bax, caspase-8, caspase-9, CFLAR, and DR5), signal transduction molecules (Akt1, p38, erbb2, and raf1), adhesion (integrins), angiogenesis (ANGPT1, FGFR2, IFNB, PDGFA, TNF, and VEGF), and invasion and metastasis (KISS1 and Timp1). The high levels of IFNB induced by CPT-11 (11-fold), which were absent in cells transfected with IRF-5 siRNA, served as a positive control for IRF-5–dependent gene expression, which was confirmed by RT-PCR (data not shown). Although high levels of p21 were detected in CPT-11–treated cells, most likely due to the activation of p53, significantly lower but detectable levels were observed in the IRF-5 knockdown, thus confirming a role for IRF-5 in p21 gene expression (3). Identical experiments were done with HCT116 cells transfected with scrambled siRNA or LacZ control siRNA and treated with CPT-11; gene expression profiles were identical to those obtained from untransfected and CPT-11–treated cells (data not shown), indicating specificity for the targeted IRF-5 siRNA.


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  		Table 1. CPT-11 induces both IRF-5 and p53 target genes

 
DNA damage signaling induces IFN regulatory factor-5 phosphorylation and activation. IRF-1 and IRF-5 are both under the transcriptional control of the JAK/STAT signaling pathway in virus-infected or IFN-treated cells (27). IRF-1 is thought to undergo posttranslational modification to activate IFN-stimulated genes (33), and IFN-{gamma}–induced tyrosine phosphorylation of IRF-1 leads to activation (34). Recently, it was shown that IRF-1 expression (mRNA and protein) and protein stability were controlled by an ATM signaling pathway in response to irradiation (9). Whereas IRF-5 is phosphorylated and activated by viral infection (21, 22) or by stimulation with the toll-like receptor-7/8 inducer resiquimod (R-848; ref. 25), the mechanism by which genotoxic stress induces IRF-5 and the signaling components upstream is currently not known. To address whether the induction of IRF-5 transcription is sufficient to induce cell death and apoptosis, or alternatively, the IRF-5 polypeptide is posttranscriptionally activated through DNA damage–induced signaling, we have examined DNA damage–induced phosphorylation of IRF-5. Although DNA damage up-regulated IRF-5 transcription (Fig. 3) with a concomitant increase in protein (Fig. 4B), it is unlikely that an increase in transcript and protein levels is sufficient for the observed effects because overexpression of IRF-5 in either p53-proficient (data not shown) or p53-deficient (Fig. 1E) cells was unable to induce spontaneous apoptosis. We examined IRF-5 phosphorylation in untreated and CPT-11–treated p53+/+ (data not shown) and p53–/– whole cell lysates by immunoprecipitation with antibodies that recognize phospho-serine, phospho-tyrosine, or phospho-threonine residues. Results in Fig. 5A show that CPT-11 induced IRF-5 phosphorylation in p53-deficient cells as early as 3 hours posttreatment on serine and threonine residues but not on tyrosine. In agreement with previous findings (21), low levels of constitutively phosphorylated IRF-5 were detected in untreated cells at serine and tyrosine residues (Fig. 5A, lanes 0). The presence of two distinct bands after immunoprecipitation with phospho-tyrosine antibodies most likely represents two distinct IRF-5 phosphorylation species, both of which disappeared after treatment with CPT-11, indicating that these constitutively phosphorylated residues may be involved in the inhibition of IRF-5 activity. Although serine phosphorylation of IRF-5 was also detected by immunoprecipitation in untreated cells, CPT-11 gave an increase in the slower migrating band only, suggesting that this is CPT-11–activated/phosphorylated IRF-5. Of interest, this slower migrating band began to disappear at 6 hours of treatment with CPT-11 and reappeared after treatment for 16 hours, indicating that the IRF-5 protein may undergo two phases of phosphorylation/activation. Unlike the phosphorylation observed at serine and tyrosine residues of IRF-5, phosphorylation on threonine residues was observed only after treatment with CPT-11, occurring as early as 3 hours poststimulation and increasing over the indicated times. These results suggest a complex pattern of IRF-5 activation/phosphorylation by CPT-11.



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  		Figure 5. IRF-5 is activated by CPT-11 in p53-proficient and p53-deficient cells. A, IRF-5 is phosphorylated on serine and threonine residues after treatment of HCT116 p53–/– cells with CPT–11. Cells were left untreated (0) or treated with CPT-11 for 3, 6, or 16 hours. Whole cell lysates (350 µg) were immunoprecipitated with phospho-serine, phospho-threonine, or phospho-tyrosine antibodies, resolved by SDS-PAGE, transferred onto Immobilon-P polyvinylidene difluoride membrane, and probed with IRF-5 antibodies. The two bands represent IRF-5–phosphorylated species. B, IRF-5 phosphorylation by CPT-11 is distinct compared with virus-mediated phosphorylation. p53–/– cells were treated with CPT-11 or infected with Newcastle disease virus over the time course indicated and 250 µg of whole cell lysates were immunoprecipitated with antibodies specific for IRF-5–phosphorylated Ser-427 and Ser-430. Twenty-microgram whole cell lysate is shown as input. Proteins were resolved by 12% SDS-PAGE, transferred, and probed with IRF-5 antibodies. C, subcellular localization of gfp-IRF-5 is altered by CPT-11 over the indicated time intervals.

 
We have previously shown that two serine residues [Ser-477/Ser-480 corresponding to IRF-5 V1 (accession no. U51127) or Ser-427/Ser-430 corresponding to IRF-5 V3/4 (accession no. AY504946/AY504947)] in the COOH terminus of IRF-5 play critical roles in the virus-induced activation of IRF-5 (22). We had generated phospho-specific IRF-5 antibodies that recognize these phosphorylated serines and have used them here for immunoprecipitation experiments to determine whether CPT-11–induced phosphorylation of IRF-5 occurs at identical residues. p53–/– cells were treated with CPT-11 or infected with Newcastle disease virus at the indicated time intervals and whole cell lysates were immunoprecipitated with phospho-IRF-5–specific antibodies. Whereas Newcastle disease virus induced phosphorylation of IRF-5 at Ser-427 and Ser-430, phosphorylation was not detected in CPT-11–treated cells, indicating that CPT-11–induced IRF-5 phosphorylation occurs at distinct serine residues (Fig. 5B).

Because phosphorylation of IRFs often leads to an alteration in their cellular localization (21, 22, 25), we examined by fluorescent microscopy the effect of CPT-11 on gfp-IRF-5 localization in p53–/– cells. IRF-5 was detected in the cytoplasm of untreated cells and after 1-hour treatment with CPT-11, ~10% of cells showed translocation to the nucleus (Fig. 5C). At 3 hours post CPT-11 treatment, the majority of cells expressing gfp-IRF-5 showed translocation to the nucleus, which correlated well with the phosphorylation-induced activation of IRF-5 (Fig. 5A).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IFNs and the family of IRFs are multifunctional proteins with pleiotropic biological effects that trigger immunomodulatory, cell growth inhibitory, and proapoptotic activities (8, 35). IFNs have been shown to be cytotoxic for malignant cells independent of cell cycle arrest and the presence or absence of wild-type p53, or expression of Bcl-2 family members (36, 37). Whereas there are numerous reports on the usefulness of IFNs for the treatment of human cancers, the molecular basis of IFN action in cancer treatment still remains largely elusive and is thus an active area of research. A better understanding of the mechanism(s) that underlies IFN proapoptotic effects and the factors that are responsible for a lack of response to IFNs would undoubtedly lead to an improved use of IFN in malignant diseases. We have currently been studying the antitumor effects of combination IFN treatments [i.e., IFN and the chemotherapeutic drug irinotecan (CPT-11)], in addition to determining mediators of the observed growth suppression by each of these agents. Given that IFN and conventional chemotherapeutic agents initiate tumor cell apoptosis through distinct signaling pathways, combination of these approaches should lead to synergistic apoptosis activation, minimization of clinical toxicities, and reduction in the probability that tumor cells will develop resistance to therapy.

Our molecular analysis of these pathways has led to the identification of IRF-1 and IRF-5 as critical transducers of IFN- and DNA damage–mediated cell growth regulation. It has been shown that the IRF-1 signaling pathway is important for regulating cell growth as a consequence of exposure to IR and in response to DNA strand breaks. Current data suggest a critical role for the IRF-5 signaling pathway in regulating cell growth in response to DNA strand breaks caused by the topoisomerase I inhibitor CPT-11. We have shown that IRF-5 is a critical mediator of both IFN- and DNA damage–induced signaling leading to the induction of apoptosis and/or cell death (Figs. 1 and 2). Overexpression of IRF-5 protein by transient transfection or stable overexpression enhanced DNA damage–induced apoptosis, and a specific suppression of IRF-5 protein by targeted siRNA repressed apoptosis and prevented enhancer activity of IFN-ß in CPT-11–induced apoptosis. The ability of IRF-5 to elevate the sensitivity of p53-deficient cells to DNA damage–induced apoptosis was independent of alterations in cell cycle or doubling time, as we could detect no substantial change in cell cycle between p53-deficient and gfp-IRF-5/p53-deficient cells (data not shown). Furthermore, our data support a combination therapy of type I IFNs and CPT-11 for cancer treatment in humans; in addition to IFNs, IRF-5 itself may combine with CPT-11 to induce effective tumor cell death.

Whereas CPT-11 promoted p53-independent expression of IRF-1 and IRF-5 gene transcripts (Fig. 3), IRF-5, and not IRF-1, function was linked to the expression of multiple molecular determinants of mitochondrial outer membrane permeabilization (Bak and Bax) and distal death machinery (Fig. 4). Although IRF-1 did not seem to be directly involved in CPT-11–mediated proapoptotic gene expression (Fig. 4C and D), combination IRF-1 plus IRF-5 siRNAs cooperatively inhibited CPT-11–mediated apoptosis, indicating that both transcription factors are important in DNA damage signaling (Fig. 2C). A complementary analysis of CPT-11–mediated target gene expression and activation revealed that IRF-5 is more directly involved in CPT-11–induced apoptosis and cell death (Fig. 4; Table 1). Moreover, IRF-5 was capable of sensitizing both p53-proficient and p53-deficient cells to DNA damage–induced apoptosis (Fig. 1 and data not shown). Given the fact that a large majority of cancers have loss of p53 function contributing to resistance and treatment failure, combined with the observation that at least some of the growth regulatory and proapoptotic functions of p53 can be compensated for by IRF-5, strategies to up-regulate endogenous IRF-5 combined with conventional chemotherapy may prove beneficial for cancer cell growth management thereby increasing the sensitivity of p53-deficient tumors to drug-induced apoptosis.

Our analysis of DNA damage–induced signaling events identified downstream components of IRF-5 signaling to include Bax, caspase-8, and caspase-3 (Fig. 6). Examination of CPT-11–mediated signaling over the indicated time course revealed that caspase-8 mRNA and protein cleavage was attenuated by IRF-5 siRNA, revealing distinct similarities with IFN signaling. Regardless of cell type or tissue histology, induction of apoptosis by all IFN subtypes (IFN-{alpha}, -ß, and -{gamma}) has involved Fas-associated death domain (FADD)/caspase-8 signaling, activation of the caspase cascade, release of cytochrome c from mitochondria, disruption of mitochondrial potential, changes in plasma membrane symmetry, and DNA fragmentation (36, 38, 39). In general, IFN-induced death has been prevented by inhibitors of caspase-8 or caspase-3, or by dominant negative mutants of FADD (40). Results from microarray analysis of CPT-11 target gene expression revealed that IRF-5 is a critical mediator of both intrinsic and extrinsic DNA damage signaling (Fig. 6; Table 1). IFNB, caspase-8, Bax, and DR5 are all highly interrelated genes associated with both IFN and IRF-5 signaling cascades (3). By analyzing gene expression in CPT-11–treated cells transiently transfected with IRF-5 siRNA (Table 1), we identified a group of genes that were either IRF-5 independent (i.e., p53 and mdm2) or of which regulation occurred upstream of IRF-5 signaling events and were thus unaffected by IRF-5 expression levels (i.e., ATM and DNA-PK). Together, these results suggest that signaling through IRF-5 may be similar to apoptotic signaling by the IFNs.



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  		Figure 6. A model for DNA damage and IFN signaling through IRF-5.

 
Posttranslational modification, nuclear translocation, and transactivating function of IRF-5 occur after virus infection and DNA damage, yet the signaling pathways leading to its activation are quite different. The involvement of IRF-5 in DNA damage response and the recent characterization of the DNA damage–sensing machineries [ATM, ATM- and Rad3-related protein (ATR), and DNA-dependent protein kinase (DNA-PK)] as more general stress sensors suggest the possibility of cross-talk between the DNA damage and virus/IFN response pathways (41, 42). The phosphatidylinositol-3 kinase–related kinases ATR, ATM, and DNA-PK become activated in response to DNA damage and transduce signals to downstream targets, including p53 and checkpoint kinases Chk1 and Chk2. Using the SCANSITE database (43), we have identified Thr-272 of the IRF-5 V3/4 polypeptide as a potential target for phosphorylation by ATM or DNA-PK. Our data indicate a complex regulatory mechanism in which CPT-11 induced both the phosphorylation and dephosphorylation of IRF-5 (Fig. 5A). Although low levels of constitutively phosphorylated IRF-5 were detected in untreated cells by immunoprecipitation with phospho-serine and phospho-tyrosine antibodies, treatment with CPT-11 gave a further increase in serine phosphorylation that was detected by a new slower migrating band. At the same time (3 hours), phosphorylation at tyrosine residues was no longer detected. The noted decrease in serine phosphorylated IRF-5 at 6 hours posttreatment may be a result of a biphasic mechanism in which the first phase of phosphorylation likely occurs with constitutively expressed IRF-5 protein (3 hours), and the second phase (8-16 hours) occurs after the up-regulation of IRF-5 gene transcripts (8 hours) and protein by CPT-11 (8-16 hours). Alternatively, these data suggest that at least two different kinases are mediating the biphasic increase in IRF-5 phosphorylation at serine residues. In contrast, phosphorylation at threonine residues was monophasic and directly linked with CPT-11 treatment. In this case, two distinct threonine-phosphorylated IRF-5 species were detected at increasing levels over the time course examined. Using our previously generated phospho-specific IRF-5 antibodies, we determined that phosphorylation of IRF-5 by CPT-11 was distinct from virus-induced phosphorylation, supporting the idea of two distinct signaling pathways leading to IRF-5 activation. Furthermore, induction of IRF-1 or IRF-5 by CPT-11 was independent of JAK/STAT signaling as neither STAT1 nor STAT2 was phosphorylated over a time course of treatments in HCT116 cells (data not shown). The CPT-11–mediated nuclear translocation of gfp-IRF-5 occurred predominantly at 3 hours, which correlated well with the initial alterations in IRF-5 phosphorylation status.

A preliminary examination of IRF-5 phosphorylation by the phosphatidylinositol-3 kinases ATM and DNA-PK indicates that DNA-PK, and not ATM, is involved in CPT-11–induced phosphorylation of IRF-5. Phosphorylation at threonine residues was unaffected in ataxia telangiectasia (GM05849) fibroblasts, yet was significantly reduced in cells lacking catalytically active DNA-PK (M059J; data not shown). Although IRF-5 phosphorylation was detected by immunoprecipitation with phospho-threonine and phospho-serine antibodies in M059J cells (44), the levels were significantly lower than those observed in p53-deficient HCT116 or ataxia telangiectasia–deficient cells treated with CPT-11 (data not shown). Because DNA-PK is located primarily within the nucleus, it seems likely that IRF-5 must first be activated and translocated to the nucleus to be a substrate for DNA-PK phosphorylation. These results support the hypothesis that at least two kinases are involved in the DNA damage–induced phosphorylation of IRF-5. Based on these findings, we are currently examining phosphorylation of IRF-5 by DNA-PK and other downstream targets of ATM/ATR or DNA-PK. Thus, in addition to identifying IRF-5 as a critical mediator of DNA damage signaling, our results highlight a role for DNA-PK in the activation of IRF-5 during the response to genotoxic stress. Further studies are necessary to fully elucidate the molecular mechanism(s) of DNA damage–induced regulation, phosphorylation, and activation of this apparent novel IRF family member. Identification of the transcription factors responsible for regulating IRF-5 expression and the kinases involved in activating IRF-5 during the response to genotoxic stress will therefore be of great interest.


   Acknowledgments
 
Grant support: American Cancer Society grant IRG-58-005-41 and a Flight Attendant Medical Research Institute Young Clinical Scientist award (B.J. Barnes).

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.

We thank Fred Bunz and members of the Barnes Lab for their critical review of the manuscript. The phospho-specific IRF-5 antibodies were generated in collaboration with Paula Pitha. HCT116 p53+/+ and p53–/– cell lines were from the laboratory of Bert Vogelstein and Ken Kinzler (Johns Hopkins University); M059 glioblastoma cell lines were from the laboratory of Ted DeWeese (Johns Hopkins University); ataxia telangiectasia fibroblasts were from the laboratory of France Carrier (University of Maryland, Baltimore).


   Footnotes
 
1 B.J. Barnes and M.E. Mancl, unpublished data. Back

Received 2/18/05. Revised 5/10/05. Accepted 6/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Takaoka A, Hayakawa S, Yanai H, et al. Integration of interferon-{alpha}/ß signaling to p53 responses in tumor suppression and antiviral defense. Nature 2003;424:516–23.[CrossRef][Medline]
  2. Pine R, Canova A, Schindler C. Tyrosine phosphorylated p91 binds to a single element in the ISGF2/IRF-1 promoter to mediate induction by IFN {alpha} and IFN {gamma}, and is likely to autoregulate the p91 gene. EMBO J 1994;13:158–67.[Medline]
  3. Barnes BJ, Kellum MJ, Pinder KE, Frisancho JA, Pitha PM. IRF-5, a novel mediator of cell-cycle arrest and cell death. Cancer Res 2003;63:6424–31.[Abstract/Free Full Text]
  4. Mori T, Anazawa Y, Iiizumi M, Fukuda S, Nakamura Y, Arakawa H. Identification of the interferon regulatory factor 5 gene (IRF-5) as a direct target for p53. Oncogene 2002;21:2914–8.[CrossRef][Medline]
  5. Tanaka N, Ishihara M, Kitagawa M, et al. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 1994;77:829–39.[CrossRef][Medline]
  6. Tamura T, Ishihara M, Lamphier MS, et al. An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. Nature 1995;376:596–9.[CrossRef][Medline]
  7. Nozawa H, Oda E, Nakao K, et al. Loss of transcription factor IRF-1 affects tumor susceptibility in mice carrying the Ha-ras transgene or nullizygosity for p53. Genes Dev 1999;13:1240–5.[Abstract/Free Full Text]
  8. Harada H, Taniguchi T, Tanaka N. The role of interferon regulatory factors in the interferon system and cell growth control. Biochimie 1998;80:641–50.[Medline]
  9. Pamment J, Ramsey E, Kelleher M, Dornan D, Ball KL. Regulation of the IRF-1 tumor modifier during the response to genotoxic stress involves an ATM-dependent signaling pathway. Oncogene 2002;21:7776–85.[CrossRef][Medline]
  10. Fujita T, Kimura Y, Miyamoto M, Barsoumian EL, Taniguchi T. Induction of endogenous IFN-{alpha} and IFN-ß genes by a regulatory transcription factor, IRF-1. Nature 1989;337:270–2.[CrossRef][Medline]
  11. Miyamoto M, Fujita T, Kimura Y, et al. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-ß gene regulatory elements. Cell 1988;54:903–13.[CrossRef][Medline]
  12. Beretta L, Gabbay M, Berger R, Hanash SM, Sonenberg N. Expression of the protein kinase PKR in modulated by IRF-1 and is reduced in 5q-associated leukemias. Oncogene 1996;12:1593–6.[Medline]
  13. Kirchhoff S, Koromilas AE, Schaper F, Grashoff M, Sonenberg N, Hauser H. IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR. Oncogene 1995;11:439–45.[Medline]
  14. Coccia EM, Del Russo N, Stellacci E, et al. Activation and repression of the 2–5A synthetase and p21 gene promoters by IRF-1 and IRF-2. Oncogene 1999;18:2129–37.[CrossRef][Medline]
  15. Wang Q, Floyd-Smith G. Maximal induction of 2',5'-oligoadenylate synthetase in Daudi cells requires cooperation between an ISRE and two IRF-1-like elements. Gene 1998;222:83–90.[CrossRef][Medline]
  16. Tanaka N, Ishihara M, Lamphier MS, et al. Cooperation of the tumour suppressors IRF-1 and p53 in response to DNA damage. Nature 1996;382:816–8.[CrossRef][Medline]
  17. Taniguchi T, Lamphier MS, Tanaka N. IRF-1: the transcription factor linking the interferon response and oncogenesis. Biochim Biophys Acta 1997;1333:M9–17.[Medline]
  18. Bowie ML, Dietze EC, Delrow J, et al. Interferon-regulatory factor-1 is critical for tamoxifen-mediated apoptosis in human mammary epithelial cells. Oncogene 2004;23:8743–55.[CrossRef][Medline]
  19. Park SY, Seol JW, Lee YJ, et al. IFN-{gamma} enhances TRAIL-induced apoptosis through IRF-1. Eur J Biochem 2004;271:4222–8.[Medline]
  20. Clarke N, Jimenez-Lara AM, Voltz E, Gronemeyer H. Tumor suppressor IRF-1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL. EMBO J 2004;23:3051–60.[CrossRef][Medline]
  21. Barnes BJ, Moore PA, Pitha PM. Virus-specific activation of a novel interferon regulatory factor, IRF-5, results in the induction of distinct interferon {alpha} genes. J Biol Chem 2001;276:23382–90.[Abstract/Free Full Text]
  22. Barnes BJ, Kellum MJ, Field AE, Pitha PM. Multiple regulatory domains of IRF-5 control activation, cellular localization and induction of chemokines involved with T-lymphocyte trafficking. Mol Cell Biol 2002;22:5721–40.[Abstract/Free Full Text]
  23. Barnes BJ, Field AE, Pitha PM. Virus-induced heterodimer formation between IRF-5 and IRF-7 modulates assembly of the IFNA enhanceosome in vivo and transcriptional activity of IFNA genes. J Biol Chem 2003;278:6630–41.
  24. Barnes BJ, Richards J, Mancl ME, Hanash S, Beretta L, Pitha PM. Global and specific targets of IRF-5 and IRF-7 during innate response to viral infection. J Biol Chem 2004;279:45194–207.[Abstract/Free Full Text]
  25. Schoenemeyer A, Barnes BJ, Mancl ME, et al. The interferon regulatory factor, IRF5, is a central mediator of TLR7 signaling. J Biol Chem 2005;280:17005–12.[Abstract/Free Full Text]
  26. Takaoka A, Yanai H, Kondo S, et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 2005;434:243–9.[CrossRef][Medline]
  27. Mancl ME, Hu G, Sangster-Guity N, et al. Two discrete promoters regulate the alternative-spliced human interferon regulatory factor-5 variants: multiple variants with distinct cell type-specific expression, localization, regulation and function. J Biol Chem 2005;280:21078–90.[Abstract/Free Full Text]
  28. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998;282:1497–501.[Abstract/Free Full Text]
  29. Saltz L. Irinotecan-based combinations for the adjuvant treatment of stage III colon cancer. Oncology (Huntingt) 2000;12:47–50.
  30. Douillard JY. V-303 Study Group. Irinotecan and high-dose fluorouracil/leucovorin for metastatic colorectal cancer. Oncology (Huntingt) 2000;12:51–5.
  31. Ravi R, Jain AJ, Schulick RD, et al. Elimination of hepatic metastases of colon cancer cells via p53-independent cross-talk between irinotecan and Apo2L/TRAIL. Cancer Res 2004;64:9105–14.[Abstract/Free Full Text]
  32. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 2003;5:834–9.[CrossRef][Medline]
  33. Watanabe N, Sakakibara J, Hovanessein AG, Taniguchi T, Fujita T. Activation of IFN-ß element by IRF-1 requires a posttranslational event in addition to IRF-1 synthesis. Nucleic Acids Res 1991;19:4421–8.[Abstract/Free Full Text]
  34. Sharf R, Meraro D, Azriel A, et al. Phosphorylation events modulate the ability of interferon consensus sequence binding protein to interact with interferon regulatory factors to bind DNA. J Biol Chem 1997;272:9785–92.[Abstract/Free Full Text]
  35. Gresser I, Belardelli F. Endogenous type I interferons as a defense against tumors. Cytokine Growth Factor Rev 2002;13:111–8.[CrossRef][Medline]
  36. Ossina NK, Cannas A, Powers VC, et al. Interferon-{gamma} modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 1997;272:16351–7.[Abstract/Free Full Text]
  37. Thyrell L, Erickson S, Zhivotovsky B, et al. Mechanisms of Interferon-{alpha} induced apoptosis in malignant cells. Oncogene 2002;21:1251–62.[CrossRef][Medline]
  38. Chawla-Sarkar M, Leaman DW, Borden EC. Preferential induction of apoptosis by interferon (IFN)-ß compared with IFN-{alpha}2: correlation with TRAIL/Apo2L induction in melanoma cell lines. Clin Cancer Res 2001;7:1821–31.[Abstract/Free Full Text]
  39. Chen Q, Gong B, Mahmoud-Ahmed AS, et al. Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma. Blood 2001;98:2183–92.[Abstract/Free Full Text]
  40. Balachandran S, Roberts PC, Kipperman T, et al. {alpha}/ß interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway. J Virol 2000;74:1513–23.[Abstract/Free Full Text]
  41. Karpova AY, Trost M, Murray JM, Cantley LC, Howley PM. Interferon regulatory factor-3 is an in vivo target of DNA-PK. Proc Natl Acad Sci U S A 2002;99:2818–23.[Abstract/Free Full Text]
  42. Yang J, Yu Y, Hamrick HE, Duerksen-Hughes PJ. ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis 2003;24:1571–80.[Abstract/Free Full Text]
  43. Yaffe MB, Leparc GG, Lai J, Obata T, Volinia S, Cantley LC. A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nat Biotechnol 2001;19:34–53.
  44. Hoppe BS, Jensen RB, Kirchgessner CU. Complementation of the radiosensitive M059J cell line. Radiat Res 2000;153:125–30.[Medline]



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