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Role of Schlafen 2 (SLFN2) in the Generation of Interferon α-induced Growth Inhibitory Responses

Role of Schlafen 2 (SLFN2) in the Generation of Interferon α-induced Growth Inhibitory Responses THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 37, pp. 25051–25064, September 11, 2009 Printed in the U.S.A. Role of Schlafen 2 (SLFN2) in the Generation of Interferon -induced Growth Inhibitory Responses Received for publication, June 5, 2009, and in revised form, July 9, 2009 Published, JBC Papers in Press, July 10, 2009, DOI 10.1074/jbc.M109.030445 ‡ ‡ ‡ ‡ § Efstratios Katsoulidis , Nathalie Carayol , Jennifer Woodard , Iwona Konieczna , Beata Majchrzak-Kita , ‡ ‡ ‡ § ‡1 Alison Jordan , Antonella Sassano , Elizabeth A. Eklund , Eleanor N. Fish , and Leonidas C. Platanias From the Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Northwestern University Medical School and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60611 and the Division of Cell and Molecular Biology, Toronto Research Institute, University Health Network and Department of Immunology, University of Toronto, Toronto, Ontario M5G 2M1, Canada The precise STAT-regulated gene targets that inhibit cell cades that are activated in an IFN-dependent manner have been growth and generate the antitumor effects of Type I interferons defined. The Jak-STAT pathway is the most important pathway (IFNs) remain unknown. We provide evidence that Type I IFNs in the regulation of IFN-inducible gene transcription and prob- regulate expression of Schlafens (SLFNs), a group of genes ably the best studied and characterized IFN-regulated signal- involved in the control of cell cycle progression and growth ing pathway to date (reviewed in Refs. 2 and 5–7). Beyond the inhibitory responses. Using cells with targeted disruption of dif- Jak-STAT pathway, other highly relevant cellular cascades in ferent STAT proteins and/or the p38 MAP kinase, we demon- IFN signaling are MAP kinase pathways (8–13) that control strate that the IFN-dependent expression of distinct Schlafen auxiliary signals for optimal gene transcription and Akt/mTOR genes is differentially regulated by STAT complexes and the p38 pathways that promote mRNA translation of IFN-stimulated MAP kinase pathway. We also provide evidence for a key func- genes (ISGs) (14–18). An emerging model for the production of tional role of a member of the SLFN family, SLFN2, in the induc- Type I IFN-inducible gene products involves transcriptional tion of the growth-suppressive effects of IFNs. This is shown in regulation of ISGs by Jak-STAT pathways, immediately fol- studies demonstrating that knockdown of SLFN2 enhances lowed by mRNA translation of such transcripts in an mTOR/ hematopoietic progenitor colony formation and reverses the 4EBP1-dependent manner (17, 18). growth-suppressive effects of IFN on normal hematopoietic The identification and definition of Type I IFN receptor- progenitors. Importantly, NIH3T3 or L929 cells with stable generated signals that promote transcription and mRNA trans- knockdown of SLFN2 form more colonies in soft agar, implicat- lation of target genes has provided critical information of how ing this protein in the regulation of anchorage-independent early signals at the receptor level ultimately translate to Type I growth. Altogether, our data implicate SLFN2 as a negative reg- IFN responses. A remaining challenge in the IFN signaling field ulator of the metastatic and growth potential of malignant cells is the identification of specific genes or groups of genes that and strongly suggest a role for the SLFN family of proteins in the specifically account for the induction of the diverse biological generation of the antiproliferative effects of Type I IFNs. responses of IFNs. Various proteins that are involved in the generation of the antiviral effects of IFNs have been identified over the years (19). However, very little is known on ISG prod- Type I interferons (IFNs) are potent inhibitors of cell growth ucts that participate in the generation of IFN-dependent anti- of both normal and malignant cells in vitro and in vivo and play proliferative responses. In fact, the key IFN-inducible gene critical roles in the immune surveillance against cancer (1–4). products that mediate growth inhibitory responses in different The potent antitumor properties of Type I IFNs have prompted cell types remain largely unknown. extensive efforts over the years to understand the mechanisms The Schlafen (SLFN) (from the German word schlafen or by which these cytokines generate signals and induce biological sleeping) family of proteins includes several members that have responses. Key events elicited during engagement of the Type I previously been shown to control cell cycle progression and IFN-receptor have been identified, and major signaling cas- growth arrest (20–26). These proteins contain a common N-terminal (AAA) domain that is involved in GTP/ATP bind- ing (20, 22), whereas a subgroup of these proteins, the long * This work was supported, in whole or in part, by National Institutes of Health Grants CA100579, CA77816, and CA121192. This work was also supported SLFNs, have motifs found in members of Superfamily I of by a Merit Review Grant from the Department of Veterans Affairs and by DNA/RNA helicases (21). There is evidence that Schlafen pro- Canadian Institutes of Health Research Grant MOP 15094. teins promote growth inhibitory responses (20) and modulate To whom correspondence should be addressed: Robert H. Lurie Compre- hensive Cancer Center, Northwestern University Medical School, 710 cell cycle progression by inhibiting cyclin D1 (22). Although North Fairbanks St., Olson 8250, Chicago, IL 60611. Tel.: 312-503-4267; Fax: limited studies have been conducted on the roles of distinct 312-908-1372; E-mail: l-platanias @northwestern.edu. Schlafen group members on the regulation of cellular func- The abbreviations used are: IFN, interferon; STAT, signal transducers and activators of transcription; MAP, mitogen-activated protein; ISG, IFN-stim- tions, there is emerging evidence indicating a potentially ulated gene; MEF, mouse embryonic fibroblast; GAPDH, glyceraldehyde-3- important role for these proteins in the control of cell cycle phosphate dehydrogenase; siRNA, small interfering RNA; Ctrl, control; progression. Regardless, very little is known on the potential shRNA, small hairpin RNA; MAPK, MAP kinase; RT, reverse transcription; SLFN, Schlafen. involvement of SLFN genes and their products in the induction SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25051 This is an Open Access article under the CC BY license. Role of SLFN2 in the Generation of IFN Responses of antiproliferative responses induced by IFNs or other growth- indicated times or were left untreated. Immunoprecipitations suppressive cytokines. and immunoblotting using an ECL method were performed as In the present study we examined the induction of expres- previously described (12, 30). sion of various mouse SLFN family members during treatment Antiviral Assays—The antiviral effects of IFN were deter- of sensitive cells with IFN. Our data demonstrate that SLFN1 mined using standard methodologies as in previous work (13), and SLFN2 (group I), SLFN3 (group II), as well as SLFN5 and using encephalomyocarditis virus as the challenge virus. SLFN8 (group III) are all genes inducible by treatment of sensi- Mobility Shift Assays—Actively growing cells were treated tive cells with mouse IFN. Using defined knock-out cells for with 10 IU/ml mouse IFN for 15 min. Equal amounts of different STAT proteins and/or the p38 MAP kinase, we pro- nuclear extracts from untreated or IFN-treated cells were ana- vide evidence for differential regulation of distinct SLFN mem- lyzed using electrophoretic mobility shift assays with oligonu- bers by different STAT complexes and the p38 MAP kinase. In cleotides to detect SIF or ISGF3 complexes, as in our previous other studies we provide evidence that knockdown of SLFN2 studies (33, 34). enhances murine hematopoietic progenitor colony formation siRNA Transfection and Generation of Stable SLFN2 Knock- and reverses the growth-suppressive effects of IFN and IFN down Cells—Transient knockdown of SLFN2 was performed on normal hematopoiesis. In addition, our data show that using either SLFN2 ON-TARGETplus SMARTpool siRNA NIH3T3 and L929 fibroblast cells with stable knockdown of (SLFN2 siRNA1) and nontargeting control pool siRNA (Ctrl SLFN2 form more colonies in soft agar compared with control siRNA1) (Thermo Fisher Scientific, Waltham, MA) or a cells, implicating this member of the SLFN family of proteins in Silencer select SLFN2 siRNA pool (SLFN2 siRNA) and a the regulation of anchorage-independent growth. Altogether, Silencer select control nontargeting siRNA (Ctrl siRNA2) our results indicate that SLFN2 acts as a negative regulator of (Applied Biosystems, Foster City, CA). The siRNA transfection the metastatic and growth potential of malignant cells, and it is reagent TransIT-TKO was used according to the manufactur- an effector element in the generation of Type I IFN-induced er’s instructions (Mirus Bio Corporation, Madison, WI). For antiproliferative responses. the generation of stable SLFN2 knockdown NIH3T3 and L929 cells, a commercially available system from Clontech MATERIALS AND METHODS was used. Briefly, SLFN2 ON-TARGETplus SMARTpool Cells Lines and Antibodies—NIH3T3 and L929 cells were siRNA and control scrambled sequences were used as tem- grown in Dulbecco’s modified Eagle’s medium, supplemented plates in the Clontech shRNA sequence designer tool for with 10% fetal calf serum and antibiotics. Immortalized mouse Clontech pSIREN vectors. Plasmids were sequenced to verify embryonic fibroblasts (MEFs) from p38 knock-out mice (27) the presence of siRNA encoding insert and then used for were kindly provided from Dr. Angel Nebreda (CNIO (Spanish retroviral infection of NIH3T3 and L929 cells. Infected pSI- National Cancer Center), Madrid, Spain). Immortalized REN-shRNA expressing cells were green fluorescent and STAT1 knock-out (28) and STAT3 knock-out (29) MEFs were were selected by flow cytometry. generously provided by Dr. David Levy (New York University, Cell Proliferation Assays—Cell proliferation assays using the New York, NY). In the figures, STAT3 WT refers to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro- flox/ STAT3 MEFs (29), whereas STAT3 KO MEFs refers to mide method were performed as in our previous studies MEFs resulting from deletion of exons 16–21 of STAT3 by (35, 36). infection with a retrovirus encoding Cre recombinase (29). The Hematopoietic Progenitor Cell Assays and Soft Agar Assays— different MEFs were cultured in Dulbecco’s modified Eagle’s Mouse hematopoietic progenitor colony formation was medium supplemented with 10% fetal calf serum and antibiot- assessed as previously described (12, 37). Colony formation ics. A custom-made polyclonal antibody against the N-terminal assays were performed using Sca1 cells isolated from mouse region (amino acids 1–14) of mouse SLFN2 was produced and bone marrow stem cells according to the manufacturer’s purified via New England Peptide LLC (Gardner, MA). Anti- instructions (MACS kit, Miltenyi Biotec Inc., CA). The cells bodies against Cyclin D1, Cyclin D3, CDK 4, CDK 6, p15 INK, were plated in methocult methylcellulose media (Stemcell and p27 KIP were obtained from Cell Signaling Technology Technologies, Seattle, WA) in the presence or absence of 10 (Danvers, MA). An antibody against glyceraldehyde-3-phos- IU/ml IFN, and colony formation was assessed after 7 days of phate dehydrogenase (GAPDH) was obtained from Chemicon culture. Anchorage-independent growth was assessed in soft International (Temecula, CA). An antibody against Lamin A was agar assays in duplicate, carried out essentially as previously purchased from Santa Cruz Biotechnology (Santa Cruz, CA). described (38). Briefly, the cells were suspended in 0.3% top Cell Lysis, Isolation of Nuclear and Cytosolic Fractions, agar over a bottom layer of 0.5% agar in 6-well plates. The solid- Immunoprecipitations, and Immunoblotting—The cells were ified soft agar was overlaid with Dulbecco’s modified Eagle’s lysed in phosphorylation lysis buffer as described in our previ- medium containing 10% fetal bovine serum and antibiotics. ous studies (12, 30). For the detection of IFN-dependent SLFN2 The medium was changed every 4–5 days. The colonies were translocation, the cells were treated with 10 IU/ml IFN for scored after 11 days (NIH3T3 cells) or 8 days (L929 cells) of the indicated times or were left untreated. Nuclear and cytoso- culture. lic fractions were isolated using the Pierce NE-PER kit accord- mRNA Isolation and Real Time PCR Probes and Primers— ing to the manufacturer’s instructions (Thermo Fisher Scien- Cells were treated with 5 10 IU/ml of IFN for the indicated tific, Waltham, MA). For the detection of SLFN2 protein times. Isolation, purification of mRNA, and conversion into expression, cells were treated with 1.5 10 IU/ml IFN for the cDNA was performed using the respective kits and oligo(dT)s 25052 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 1. IFN-inducible expression of SLFN family members. A–E, NIH3T3 cells were treated with IFN for3or6hor left untreated as indicated. Total RNA was subsequently isolated, and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5(D), and SLFN8 (E) was analyzed by real time RT-PCR, using specific primers and GAPDH as an internal control. The data are expressed as fold increase over control untreated samples and represent the means  S.E. of several experiments for SLFN1 (n 3), SLFN2 (n 7), SLFN3 (n 6), SLFN5 (n 4), and SLFN8 (n 5). F, NIH3T3 cells were treated with IFN for 48 h, and after cell lysis, the proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN2 antibody. G, NIH3T3 cells were treated with mouse IFN, as indicated. Cytosolic and nuclear fractions were obtained, and the proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN2 antibody. Immunoblot- ting with antibodies against Lamin A and GAPDH was also performed to control for successful separation of nuclear and cytosolic fractions. from Qiagen according to the manufacturer’s instructions. Val- induction of mRNA expression for key members of the SLFN idated, inventoried probes and primers for real time PCR and gene family was determined. As shown in Fig. 1, mRNA expres- TaqMan PCR master mix were purchased from Applied Bio- sion for different SLFN genes was inducible at various degrees systems (Foster City, CA). The probes and primers were: in response to IFN treatment. The most pronounced induc- SLFN1, Mm00488306_m1; SLFN2, Mm 00488307_m1; SLFN3, tion was for SLFN1 (Fig. 1A), followed by SLFN5, SLFN2, and Mm00488309_g1; SLFN5, Mm00806095_m1; SLFN8, SLFN8 (Fig. 1, B, D, and E). SLFN3 was induced clearly to a Mm00824405_m1; and ISG15, Mm01705338_s1. GAPDH lesser degree than other SLFNs(4-fold), but its induction was (Mm99999915_g1) was used as an internal control. consistently seen (Fig. 1C). To better understand the regulation of SLFN proteins during RESULTS engagement of the Type I IFN receptor, we generated and used In initial studies we determined whether treatment of cells an anti-SLFN2 antibody to directly examine the expression of with IFN induces expression of different SLFN genes. NIH3T3 SLFN2 protein after IFN treatment of cells. This antibody was cells were treated with mouse IFN for different times, and the custom-generated via a commercial vendor against a conserved SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25053 Role of SLFN2 in the Generation of IFN Responses / / FIGURE 2. Differential requirement for STAT1 in IFN-inducible expression of distinct SLFN mRNAs. STAT1 and STAT1 MEFs were treated with IFN for the indicated times. Total RNA was isolated and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5 (D), and SLFN8 (E) mRNAs was determined by real time RT-PCR, after normalization for GAPDH expression. The data are expressed as fold increases over control untreated samples and represent the means  S.E. of two independent experiments for SLFN3 and SLFN5, three for SLFN8, four for SLFN1, and seven for SLFN2. 25054 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 3. Differential requirement for STAT3 in IFN-dependent expression of distinct SLFN mRNAs. The indicated MEFs were treated with IFN for the indicated times. Total RNA was isolated and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5 (D), and SLFN8 (E) mRNAs was determined by real time RT-PCR, after normalization for GAPDH expression. The data are expressed as fold increases over control untreated samples and represent the means S.E. of three independent experiments for SLFN1, SLFN2, and SLFN5 and six for SLFN8. region in the N terminus of the protein and detects a single cells with IFN resulted in up-regulation of the expression of band at44 kDa, which is consistent with the predicted molec- the protein (Fig. 1F). We also examined the subcellular local- ular mass of SLFN2. As shown in Fig. 1F, base-line expression of ization of the protein. In a previous study, it was shown that SLFN2 in NIH3T3 cells was clearly detectable, but treatment of overexpressed FLAG-tagged SLFN2 in HEK-293T cells is SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25055 Role of SLFN2 in the Generation of IFN Responses / / FIGURE 4. Role of p38 MAPK in the regulation of expression of SLFN genes. p38 and p38 MEFs were treated with IFN for the indicated times. Total RNA was isolated and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5 (D), and SLFN8 (E) mRNAs was determined by real time RT-PCR, after normalization for GAPDH expression. The data are expressed as fold increases over control untreated samples and represent the means  S.E. of three independent experiments for SLFN2, four for SLFN1 and SLFN3, and five for SLFN5 and SLFN8. 25056 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 5. SLFN2 controls hematopoietic progenitor colony formation and promotes the growth-suppressive effects of IFNs on primitive hematopoi- etic precursors. A, to test whether the available siRNAs were efficient and selective for the knockdown of SLFN2 in murine cells, NIH3T3 cells were transfected with control siRNAs or siRNAs selectively targeting SLFN2, and expression of SLFN2 or SLFN3 mRNAs was subsequently examined by real time RT-PCR. Two different pools of SLFN2 siRNA (siRNA1 and siRNA2) and control siRNA (Ctrl siRNA1 and Ctrl siRNA2) were used. The data are presented as percentages of expression in control siRNA transfected cells and represent the means  S.E. of three experiments. B, Sca1 derived, murine hematopoietic progenitor cells were transfected with control siRNA or SLFN2-siRNA, and hematopoietic colony progenitor was assessed in clonogenic assays in methylcellulose. Represent- ative plates are shown. C–D. Sca1 stem cells were isolated from murine bone marrows and plated in methylcellulose in the presence or absence of IFN (C and D). The cells were either not transfected or were transfected with the control nontargeting siRNAs or SLFN2-targeting siRNAs shown in A. Colony formation (colony forming units) of primitive hematopoietic precursors was assessed at day 7 of culture. The data are expressed as percentages of control untransfected cells colony formation and represent means S.E. of five (C) or four (D) independent experiments. Paired t test analysis demonstrated a p value of 0.0004 for IFN-treated Ctrl siRNA1 versus SLFN2 siRNA1 transfected cells (C) and a p value of 0.007 for IFN-treated Ctrl siRNA2 versus SLFN2 siRNA2 transfected cells (D). exclusively expressed in the cytoplasm (25). However, FLAG the effect ranged from a partial impairment (SLFN3) to com- tagging could theoretically interfere with the structural proper- pletely defective transcription (SLFN1, 2, 5, and 8) (Fig. 2). Sim- ties and localization of the protein, and the potential transloca- ilarly, IFN-inducible SLFN expression was examined in tion of endogenous SLFN2 in response to cytokine treatment STAT3 knock-out MEF cells. The induction of expression of has not been known. In studies in which the localization of the SLFN1, SLFN2, SLFN3, and SLFN8 genes was decreased in endogenous protein was directly determined using the newly STAT3 knock-out cells although not abrogated (Fig. 3, A–C). generated anti-SLFN2 antibody, we found that endogenous The expression of SLFN5 was completely STAT3-independent, SLFN2 is exclusively expressed in the cytoplasm, and IFN and in fact, SLFN5 expression was enhanced in STAT3 knock- treatment does not induce its translocation to the nucleus out cells (Fig. 3D). (Fig. 1G). p38 MAPK-activated signaling cascades play important roles IFN binding to the type I IFN receptor results in activation in Type I IFN-dependent transcriptional regulation, acting as of STAT1, STAT2, and STAT3 transcription factors, which auxiliaries to STAT pathways, and their function is essential for form various homo- and/or heterodimers that can bind specific full transcriptional activation of ISGs (reviewed in Refs. 7 and sequences in the promoters of IFN inducible genes (2–7). In 39). To determine the role of p38 MAPK-mediated signals in addition, IFN-mediated gene transcription is regulated by SLFN gene expression, we used MEF cells with targeted disrup- auxiliary pathways, such as the p38 MAPK pathway (7–13). To tion of the p38 gene (27) in which we have previously shown define the roles of distinct STAT proteins and the p38 MAPK in that IFN-inducible transcription via ISRE or GAS elements is SLFN gene expression, experiments were performed using cells defective (33). IFN-dependent mRNA expression for SLFN1, with targeted disruption of STAT1, STAT3,orp38 genes. In SLFN2, and, to a lesser degree, SLFN3 was suppressed in the / / initial studies, STAT1 MEFs and STAT1 parental MEFs absence of p38 MAPK (Fig. 4, A–C). On the other hand, the were treated with mouse IFN, and mRNA expression for group III schlafen genes, SLFN5 and SLFN8, were induced by SLFN1, SLFN2, SLFN3, SLFN5, and SLFN8 was determined. IFN in a p38 MAPK-independent manner (Fig. 4, D and E), IFN-dependent expression of all SLFN genes was decreased in suggesting that p38 activity is essential for IFN-dependent STAT1 knock-out MEFs compared with parental MEFs, and expression of group I and II but not group III Schlafen genes. SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25057 Role of SLFN2 in the Generation of IFN Responses FIGURE 6. Stable knockdown of SLFN2 enhances cell proliferation and impairs IFN-dependent growth inhibitory responses but has no effects on the generation of antiviral responses. A, expression of SLFN2 or SLFN1 mRNAs in pSIREN Zsgreen-SLFN2 siRNA and pSIREN Zsgreen control-siRNA NIH3T3 cells was determined by real time RT-PCR using specific primers and GAPDH as an internal control. The data are presented as percentages of expression in pSIREN Zsgreen control-siRNA cells and represent the means  S.E. of three experiments. B, total cell lysates from pSIREN Zsgreen SLFN2-siRNA or pSIREN Zsgreen control-siRNA NIH3T3 cells were resolved by SDS-PAGE and immunoblotted sequentially with anti-SLFN2 or anti-GAPDH antibodies. C, equal numbers of pSIREN Zsgreen SLFN2-siRNA or pSIREN Zsgreen control-siRNA NIH3T3 cells were plated and were left untreated or were treated with the indicated doses of mouse IFN. After 5–7 days cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays. A representative experiment is shown in the left panel. The means S.E. of three experiments, including the one shown in the picture on the left, are shown on the right panel. D and E, pSIREN Zsgreen SLFN2 siRNA or pSIREN Zsgreen control-siRNA NIH3T3 cells were treated with IFN for 15 min, as indicated. Nuclear extracts were reacted with 40,000 cpm of P-labeled ISRE (D) or SIE (E) oligonucleotides, and complexes were resolved by native gel electrophoresis and visualized by autoradiography. The migration of the different STAT complexes is indicated by arrows. F, wild-type NIH3T3 cells, pSIREN Zsgreen SLFN2 siRNA NIH3T3 cells, or pSIREN Zsgreen control-siRNA NIH3T3 cells were treated with IFN for the indicated times. Expression of Isg15 mRNA was determined by real time RT-PCR using GAPDH as an internal control. The data are expressed as the means S.E. of three experiments. G, wild-type NIH3T3 cells, pSIREN Zsgreen SLFN2-siRNA NIH3T3 cells, or pSIREN Zsgreen control-siRNA NIH3T3 cells were incubated in triplicate, in the presence or absence of the indicated concentrations of IFN. The cells were subsequently challenged with encephalomyocarditis virus (EMCV), and cytopathic effects were quantified 24 h later. The data are expressed as percent- ages of protection from the cytopathic effects of encephalomyocarditis virus. A representative of three independent experiments is shown. It is well known that Type I and II IFNs are potent regulators as expected, treatment of cells with IFN (Fig. 5, C and D)or of normal and leukemic hematopoiesis and inhibit the growth IFN (data not shown) resulted in suppression of hematopoi- of primitive hematopoietic precursors in vitro and in vivo (4, 39, etic progenitor colony formation compared with untreated 40). It has been also established that activation of the p38 MAP cells, although the suppressive effects of IFN were much less kinase pathway is required for the generation of the myelosup- noticeable in cells in which SLFN2 was knocked down (Fig. 5, C pressive effects of IFNs on both normal and leukemic progeni- and D). Thus, SLFN2 participates in the control of normal tors (12, 13). Because SLFN2 is induced by IFNs and its expres- hematopoiesis and the generation of the myelossuppressive sion is regulated via both STAT and p38 MAPK pathways, we effects of IFNs, suggesting that this protein may be an effector examined whether this protein plays a role in the generation of in the regulation of p38-mediated hematopoietic suppression. the myelosuppressive effects of IFN. In initial experiments, To further analyze the functional relevance of SLFN2 in cell two different specific siRNAs (Fig. 5A) were used to knock growth regulation and its role in the generation of IFN down SLFN2 expression in murine bone marrow-derived responses in other cell types, we generated stable SLFN2 knock- Sca1 stem cells. Primitive progenitor colony formation was down NIH3T3 cells via expression of shRNA-targeting SLFN2 subsequently assessed in clonogenic assays in methylcellulose. using the pSIREN Zsgreen retroviral system. SLFN2 expression Knockdown of SLFN2 in normal hematopoietic progenitors was selectively knocked down in NIH3T3 cells (Fig. 6, A and B). resulted in increased hematopoietic colony formation (Fig. 5, Cells in which SLFN2 was knocked down exhibited enhanced B–D), suggesting that this protein plays a critical role in the proliferation compared with their control counterparts (Fig. control of normal hematopoietic progenitor cell growth. Also, 6C). IFN treatment resulted in dose-dependent growth sup- 25058 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 6 —continued pression in both NIH3T3 pSIREN Zsgreen Ctrl siRNA and appears to be impairing IFN-dependent cell cycle arrest but NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells (Fig. 6C). How- not IFN-inducible gene transcription or generation of antiviral ever, in NIH3T3 cells in which SLFN2 was knocked down, responses. IFN-induced antiproliferative responses were clearly To examine whether SLFN2 plays a role in the control of decreased compared with cells expressing SLFN2 (Fig. 6C), anchorage-independent growth, we assayed transduced indicating that SLFN2 participates in the generation of the NIH3T3 cells for colony formation in soft agar (41). Colony growth inhibitory effects of IFN. On the other hand, SLFN2 formation was clearly increased in NIH3T3 pSIREN Zsgreen knockdown had no effect on IFN-dependent formation of SLFN2-siRNA cells as compared with NIH3T3 pSIREN STAT-containing DNA-binding complexes (Fig. 6, D and E). Zsgreen Ctrl siRNA cells (Fig. 7, A and B). Notably, the colonies Similarly, IFN-dependent Isg15 gene transcription (Fig. 6F)or from NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells were con- generation of IFN-induced antiviral responses (Fig. 6G) were sistently larger (Fig. 7A), and the numbers of colonies were not affected by SLFN2 knockdown. Thus, targeting SLFN2 increased (Fig. 7B) as compared with NIH3T3 pSIREN Zsgreen SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25059 Role of SLFN2 in the Generation of IFN Responses FIGURE 7. Effects of SLFN2 knockdown on anchorage-independent growth. A, equal numbers of NIH3T3 pSIREN Zsgreen control-siRNA and NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells were plated in a soft agar assay system. Colony formation was analyzed after 11 days of culture. Representative areas of the soft agar plates for NIH3T3 pSIREN Zsgreen control-siRNA and NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells are shown. B, colonies were counted, and the results were expressed as percentages of control of NIH3T3 pSIREN Zsgreen control-siRNA-derived colonies. The data shown represent the means  S.E. of three inde- pendent experiments, including the one shown in A. Paired t test analysis showed a p value of 0.01. although additional mechanisms may be involved, these findings sug- gest that SLFN2 inhibits cell growth and colony formation in part via suppression of cyclin D1 and up-regulation of the CDK inhibitor p15 INK. To definitively establish the role of SLFN2 in the generation of IFN responses and anchorage-indepen- FIGURE 8. SLFN2 knockdown in NIH3T3 cells modulates expression of Cyclin D1 and p15 INK cell cycle dent growth in nonhematopoietic regulators. NIH3T3 pSIREN Zsgreen control-siRNA and NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells were syn- cells, we stably knocked down chronized via serum starvation. After re-entry of cells into the cell cycle by the addition of serum, the cells were collected at the indicated time points. Total cell lysates were resolved by SDS-PAGE and immunoblotted with SLFN2 in another murine fibroblast antibodies against Cyclin D1 (A) or p15 INK (B), as indicated. cell line, L929. Initially, we exam- ined the IFN-inducible expression Ctrl siRNA cells. Taken altogether, these data for the first time of SLFN2 in L929 cells. The cells were treated with mouse IFN implicate SLFN2 in the regulation of anchorage-independent for different times, and the induction of mRNA and protein growth. expression was analyzed. As expected, both SLFN2 mRNA (Fig. In subsequent studies, we sought to obtain information on 9A) and protein (Fig. 9B) expression were up-regulated in the mechanisms by which SLFN2 regulates anchorage-inde- response to IFN treatment. We then generated stable SLFN2 pendent cell growth and blocks cell proliferation. Initially, we knockdown L929 cells via expression of shRNA-targeting examined the effects of SLFN2 knockdown on the expression of SLFN2 using the same pSIREN Zsgreen retroviral system we various key cell cycle regulators. We compared the levels of utilized before to knock down SLFN2 in NIH3T3 cells. Green expression of Cyclin D1, Cyclin D3, CDK4, CDK6, and the CDK fluorescent L929 pSIREN Zsgreen cells were selected after ret- inhibitors p27 KIP1 and p15 INK in serum-starved and cycling roviral transfection and analyzed for SLFN2 expression. As NIH3T3 control cells or NIH3T3 cells in which SLFN2 was shown in Fig. 9 (C and D), stable SLFN2 expression was selec- knocked down. As shown in Fig. 8A, stable SLFN2 knockdown tively knocked down in L929 pSIREN Zsgreen SLFN2-siRNA in NIH3T3 cells resulted in higher basal Cyclin D1 levels of cells compared with L929 pSIREN Zsgreen Ctrl siRNA cells. expression than control cells, whereas Cyclin D1 levels were We next analyzed IFN-dependent Isg15 gene transcription also consistently higher in cycling SLFN2 knockdown cells (Fig. 9E) in SLFN2 stable knockdown L929 cells, as well as the compared with control cells (Fig. 8A). On the other hand, effects of stable SLFN2 knockdown on IFN-induced antipro- Cyclin D3, as well as CDK4 and CDK6, levels were not signifi- liferative responses (Fig. 9F). Consistent with the results cantly altered in cells in which SLFN2 was knocked down across obtained with NIH3T3 cells, L929 cells with stable SLFN2 the time points analyzed (data not shown). When the levels of knockdown showed enhanced proliferation and were less sen- expression of the CDK inhibitors p27 KIP1 and p15 INK were sitive to the suppressive effects of IFN compared with their assessed, we noticed that unlike p27 expression, which was not control counterparts (Fig. 9F), whereas IFN-dependent Isg15 consistently altered (data not shown), p15 INK levels were gene transcription was unaltered (Fig. 9E). clearly lower in resting and cycling NIH3T3 pSIREN Zsgreen SLFN2-siRNA transfected cells, compared with NIH3T3 pSI- We also determined whether SLFN2 knockdown in L929 REN Zsgreen Ctrl siRNA transfected cells (Fig. 8B). Thus, cells enhances anchorage-independent growth. L929 pSI- 25060 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 9. Stable knockdown of SLFN2 enhances cell proliferation and impairs IFN-dependent growth inhibitory responses. A, L929 cells were treated with IFN for3or6hor left untreated as indicated. Total RNA was subsequently isolated, and the expression of SLFN2 mRNA was analyzed by real time RT-PCR, using specific primers for SLFN2 and GAPDH as an internal control. The data are expressed as fold increases over untreated samples and represent the means S.E. of four independent experiments. B, L929 cells were either left untreated or were treated with IFN for 24 or 48 h, as indicated. After cell lysis, the proteins were resolved by SDS-PAGE and immunoblotted with anti-SLFN2 or anti-GAPDH antibodies, as indicated. C, expression of SLFN2 or SLFN3 mRNAs in L929 Zsgreen-Ctrl siRNA and pSIREN Zsgreen SLFN2-siRNA in L929 cells was determined by real time RT-PCR using specific primers and GAPDH as an internal control. The data are presented as percentages of expression in pSIREN Zsgreen control-siRNA cells and represent means S.E. of five experiments. D, total cell lysates from pSIREN Zsgreen SLFN2-siRNA or pSIREN Zsgreen control-siRNA L929 cells were resolved by SDS-PAGE and immunoblotted sequentially with anti-SLFN2 or anti-GAPDH antibodies. E, wild-type L929 cells, pSIREN Zsgreen SLFN2 siRNA L929 cells or pSIREN Zsgreen control-siRNA L929 cells were treated with IFN for the indicated times. Expression of Isg15 mRNA was determined by real time RT-PCR using GAPDH as an internal control. The data are expressed as fold increases over control untreated cells and represent the means  S.E. of four experiments. F, equal numbers of L929-pSIREN Zsgreen SLFN2-siRNA or L929-pSIREN Zsgreen control-siRNA cells were either left untreated or treated with the indicated doses of mouse IFN for 5 days, and cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays. The means  S.E. of three experi- ments are shown. SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25061 Role of SLFN2 in the Generation of IFN Responses FIGURE 10. Effects of SLFN2 knockdown on anchorage-independent growth of L929 cells. A, equal numbers of L929 pSIREN Zsgreen control-siRNA and L929 pSIREN Zsgreen SLFN2-siRNA cells were plated in a soft agar assay system. Colony formation was analyzed after 8 days of culture. Representative areas of the soft agar plates for L929 pSIREN Zsgreen control-siRNA and L929 pSIREN Zsgreen SLFN2-siRNA cells are shown. B, colonies were counted, and the results were expressed as percentages of control of L929 pSIREN Zsgreen control-siRNA-derived colonies. The data shown represent the means  S.E. of four independent experiments, including the one shown in A. Paired t test analysis showed a p value of 0.04. REN Zsgreen SLFN2-siRNA and L929 pSIREN Zsgreen Ctrl Superfamily I DNA/RNA helicase motif not found in group I/II siRNA cells were plated, and colony formation was deter- SLFNs, whereas the members of this group are significantly mined after 8 days of culture in soft agar. As depicted in Fig. larger proteins with molecular masses ranging between 100 10, L929 cells with stable SLFN2 knockdown showed consis- (SLFN5) and 104 kDa (SLFN8) (21). Although the roles of tently larger colonies (Fig. 10A), and there were increased members of this SLFN group remains to be established, studies numbers of colonies compared with L929 pSIREN Zsgreen with SLFN8 transgenic mice have suggested an important reg- Ctrl siRNA cells (Fig. 10B). ulatory role for this SLFN gene in T cell development and dif- ferentiation (21). Notably, different SLFN family members have DISCUSSION been shown to be induced in response to a wide variety of stim- The family of Schlafen genes was originally identified during uli, including CpG-DNA (24), the bacterial pathogens Brucella screening for growth regulatory genes that are differentially and Listeria (44), and terminal differentiation of myeloid cells expressed during lymphocyte development (20). Originally, (21), suggesting that signals from divergent stimuli converge on SLFN family members 1, 2, 3, and 4 were identified and studied SLFN family members to control cell cycle progression. (20). Initial studies had suggested that SLFN genes suppress Despite the fact that studies on the functional relevance and growth and participate in the maintenance of the quiescent biochemical activities of SLFN proteins have been very limited state of naive T lymphocytes, as shown by experiments involv- so far, the emerging evidence suggests key regulatory roles for ing ectopic expression of SLFN1, demonstrating disruption of these proteins on cell cycle progression and growth arrest. Yet thymic development (20). Subsequently, and based on very little is known on their potential involvement in the gen- sequence homology, Geserick et al. (21) identified additional eration of the suppressive effects of growth inhibitory cyto- SLFN genes (SLFN5, SLFN8, SLFN9, and SLFN10) forming a kines. Type I IFNs are probably the most prominent cytokines cluster on mouse chromosome 11 where the SLFN1–4 genes that generate growth inhibitory and antitumor effects; and are also located. these properties have over the years led to their introduction The different members of the SLFN family of proteins can be in the treatment of various leukemias and solid tumors (3). Impor- classified into three subgroups (20, 21). The first group includes tantly, although it is well established that IFNs regulate cell cycle SLFN1 and SLFN2, which encode for the smallest two SLFN progression and induce G /G cell cycle arrest, very little is 0 1 proteins, with predicted molecular masses of 37 and 42 kDa, known about the IFN-inducible proteins that mediate such respectively (20). They contain an AAA domain, found in responses. In the present study, we provide the first evidence ATPases (42), and an adjacent “SLFN box,” which is common to that IFNs regulate expression of members of the SLFN family of all SLFN proteins (21, 25). Overexpression of SLFN1 results in genes and proteins. Our data demonstrate that IFN is a potent potent growth suppression by inducing G cell cycle arrest (20) inducer of different SLFN family members, including members through inhibition of cyclin D1 expression (22). In addition, it of Group I (SLFN1 and SLFN2), Group II (SLFN3), and Group appears that accumulation of SLFN1 protein to the nucleus III (SLFN5 and SLFN8). Moreover, in work aimed to define the correlates with induction of its growth-suppressive effects (43). regulation of expression of these proteins by IFNs, we estab- The second group of SLFN proteins includes SLFN3 and lished the differential involvement of distinct IFN-activated SLFN4, which have predicted molecular masses of 58 and 68 kDa, respectively. These proteins have in their structures a STAT proteins and the p38 MAP kinase in their regulation. small sequence motif (SWA(L/V)DL) (21, 25), also shared by Our finding that members of the SLFN family of proteins are the third group. This third group of SLFN proteins contains a engaged by the Type I IFN receptor in a STAT- and/or p38 25062 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses MAPK-dependent manner provided a direct link between IFN- development of methodologies to selectively induce SLFN gene activated Jak-STAT pathways and cellular elements controlling expression may specifically promote the antitumor effects of cell cycle progression. Such a link led us to further studies IFNs in the absence of engagement of other pathways associ- aimed to define the functional relevance of the SLFN pathway ated with various IFN-inducible adverse effects. Although the in the generation of IFN responses. 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Role of Schlafen 2 (SLFN2) in the Generation of Interferon α-induced Growth Inhibitory Responses

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 37, pp. 25051–25064, September 11, 2009 Printed in the U.S.A. Role of Schlafen 2 (SLFN2) in the Generation of Interferon -induced Growth Inhibitory Responses Received for publication, June 5, 2009, and in revised form, July 9, 2009 Published, JBC Papers in Press, July 10, 2009, DOI 10.1074/jbc.M109.030445 ‡ ‡ ‡ ‡ § Efstratios Katsoulidis , Nathalie Carayol , Jennifer Woodard , Iwona Konieczna , Beata Majchrzak-Kita , ‡ ‡ ‡ § ‡1 Alison Jordan , Antonella Sassano , Elizabeth A. Eklund , Eleanor N. Fish , and Leonidas C. Platanias From the Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Northwestern University Medical School and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60611 and the Division of Cell and Molecular Biology, Toronto Research Institute, University Health Network and Department of Immunology, University of Toronto, Toronto, Ontario M5G 2M1, Canada The precise STAT-regulated gene targets that inhibit cell cades that are activated in an IFN-dependent manner have been growth and generate the antitumor effects of Type I interferons defined. The Jak-STAT pathway is the most important pathway (IFNs) remain unknown. We provide evidence that Type I IFNs in the regulation of IFN-inducible gene transcription and prob- regulate expression of Schlafens (SLFNs), a group of genes ably the best studied and characterized IFN-regulated signal- involved in the control of cell cycle progression and growth ing pathway to date (reviewed in Refs. 2 and 5–7). Beyond the inhibitory responses. Using cells with targeted disruption of dif- Jak-STAT pathway, other highly relevant cellular cascades in ferent STAT proteins and/or the p38 MAP kinase, we demon- IFN signaling are MAP kinase pathways (8–13) that control strate that the IFN-dependent expression of distinct Schlafen auxiliary signals for optimal gene transcription and Akt/mTOR genes is differentially regulated by STAT complexes and the p38 pathways that promote mRNA translation of IFN-stimulated MAP kinase pathway. We also provide evidence for a key func- genes (ISGs) (14–18). An emerging model for the production of tional role of a member of the SLFN family, SLFN2, in the induc- Type I IFN-inducible gene products involves transcriptional tion of the growth-suppressive effects of IFNs. This is shown in regulation of ISGs by Jak-STAT pathways, immediately fol- studies demonstrating that knockdown of SLFN2 enhances lowed by mRNA translation of such transcripts in an mTOR/ hematopoietic progenitor colony formation and reverses the 4EBP1-dependent manner (17, 18). growth-suppressive effects of IFN on normal hematopoietic The identification and definition of Type I IFN receptor- progenitors. Importantly, NIH3T3 or L929 cells with stable generated signals that promote transcription and mRNA trans- knockdown of SLFN2 form more colonies in soft agar, implicat- lation of target genes has provided critical information of how ing this protein in the regulation of anchorage-independent early signals at the receptor level ultimately translate to Type I growth. Altogether, our data implicate SLFN2 as a negative reg- IFN responses. A remaining challenge in the IFN signaling field ulator of the metastatic and growth potential of malignant cells is the identification of specific genes or groups of genes that and strongly suggest a role for the SLFN family of proteins in the specifically account for the induction of the diverse biological generation of the antiproliferative effects of Type I IFNs. responses of IFNs. Various proteins that are involved in the generation of the antiviral effects of IFNs have been identified over the years (19). However, very little is known on ISG prod- Type I interferons (IFNs) are potent inhibitors of cell growth ucts that participate in the generation of IFN-dependent anti- of both normal and malignant cells in vitro and in vivo and play proliferative responses. In fact, the key IFN-inducible gene critical roles in the immune surveillance against cancer (1–4). products that mediate growth inhibitory responses in different The potent antitumor properties of Type I IFNs have prompted cell types remain largely unknown. extensive efforts over the years to understand the mechanisms The Schlafen (SLFN) (from the German word schlafen or by which these cytokines generate signals and induce biological sleeping) family of proteins includes several members that have responses. Key events elicited during engagement of the Type I previously been shown to control cell cycle progression and IFN-receptor have been identified, and major signaling cas- growth arrest (20–26). These proteins contain a common N-terminal (AAA) domain that is involved in GTP/ATP bind- ing (20, 22), whereas a subgroup of these proteins, the long * This work was supported, in whole or in part, by National Institutes of Health Grants CA100579, CA77816, and CA121192. This work was also supported SLFNs, have motifs found in members of Superfamily I of by a Merit Review Grant from the Department of Veterans Affairs and by DNA/RNA helicases (21). There is evidence that Schlafen pro- Canadian Institutes of Health Research Grant MOP 15094. teins promote growth inhibitory responses (20) and modulate To whom correspondence should be addressed: Robert H. Lurie Compre- hensive Cancer Center, Northwestern University Medical School, 710 cell cycle progression by inhibiting cyclin D1 (22). Although North Fairbanks St., Olson 8250, Chicago, IL 60611. Tel.: 312-503-4267; Fax: limited studies have been conducted on the roles of distinct 312-908-1372; E-mail: l-platanias @northwestern.edu. Schlafen group members on the regulation of cellular func- The abbreviations used are: IFN, interferon; STAT, signal transducers and activators of transcription; MAP, mitogen-activated protein; ISG, IFN-stim- tions, there is emerging evidence indicating a potentially ulated gene; MEF, mouse embryonic fibroblast; GAPDH, glyceraldehyde-3- important role for these proteins in the control of cell cycle phosphate dehydrogenase; siRNA, small interfering RNA; Ctrl, control; progression. Regardless, very little is known on the potential shRNA, small hairpin RNA; MAPK, MAP kinase; RT, reverse transcription; SLFN, Schlafen. involvement of SLFN genes and their products in the induction SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25051 This is an Open Access article under the CC BY license. Role of SLFN2 in the Generation of IFN Responses of antiproliferative responses induced by IFNs or other growth- indicated times or were left untreated. Immunoprecipitations suppressive cytokines. and immunoblotting using an ECL method were performed as In the present study we examined the induction of expres- previously described (12, 30). sion of various mouse SLFN family members during treatment Antiviral Assays—The antiviral effects of IFN were deter- of sensitive cells with IFN. Our data demonstrate that SLFN1 mined using standard methodologies as in previous work (13), and SLFN2 (group I), SLFN3 (group II), as well as SLFN5 and using encephalomyocarditis virus as the challenge virus. SLFN8 (group III) are all genes inducible by treatment of sensi- Mobility Shift Assays—Actively growing cells were treated tive cells with mouse IFN. Using defined knock-out cells for with 10 IU/ml mouse IFN for 15 min. Equal amounts of different STAT proteins and/or the p38 MAP kinase, we pro- nuclear extracts from untreated or IFN-treated cells were ana- vide evidence for differential regulation of distinct SLFN mem- lyzed using electrophoretic mobility shift assays with oligonu- bers by different STAT complexes and the p38 MAP kinase. In cleotides to detect SIF or ISGF3 complexes, as in our previous other studies we provide evidence that knockdown of SLFN2 studies (33, 34). enhances murine hematopoietic progenitor colony formation siRNA Transfection and Generation of Stable SLFN2 Knock- and reverses the growth-suppressive effects of IFN and IFN down Cells—Transient knockdown of SLFN2 was performed on normal hematopoiesis. In addition, our data show that using either SLFN2 ON-TARGETplus SMARTpool siRNA NIH3T3 and L929 fibroblast cells with stable knockdown of (SLFN2 siRNA1) and nontargeting control pool siRNA (Ctrl SLFN2 form more colonies in soft agar compared with control siRNA1) (Thermo Fisher Scientific, Waltham, MA) or a cells, implicating this member of the SLFN family of proteins in Silencer select SLFN2 siRNA pool (SLFN2 siRNA) and a the regulation of anchorage-independent growth. Altogether, Silencer select control nontargeting siRNA (Ctrl siRNA2) our results indicate that SLFN2 acts as a negative regulator of (Applied Biosystems, Foster City, CA). The siRNA transfection the metastatic and growth potential of malignant cells, and it is reagent TransIT-TKO was used according to the manufactur- an effector element in the generation of Type I IFN-induced er’s instructions (Mirus Bio Corporation, Madison, WI). For antiproliferative responses. the generation of stable SLFN2 knockdown NIH3T3 and L929 cells, a commercially available system from Clontech MATERIALS AND METHODS was used. Briefly, SLFN2 ON-TARGETplus SMARTpool Cells Lines and Antibodies—NIH3T3 and L929 cells were siRNA and control scrambled sequences were used as tem- grown in Dulbecco’s modified Eagle’s medium, supplemented plates in the Clontech shRNA sequence designer tool for with 10% fetal calf serum and antibiotics. Immortalized mouse Clontech pSIREN vectors. Plasmids were sequenced to verify embryonic fibroblasts (MEFs) from p38 knock-out mice (27) the presence of siRNA encoding insert and then used for were kindly provided from Dr. Angel Nebreda (CNIO (Spanish retroviral infection of NIH3T3 and L929 cells. Infected pSI- National Cancer Center), Madrid, Spain). Immortalized REN-shRNA expressing cells were green fluorescent and STAT1 knock-out (28) and STAT3 knock-out (29) MEFs were were selected by flow cytometry. generously provided by Dr. David Levy (New York University, Cell Proliferation Assays—Cell proliferation assays using the New York, NY). In the figures, STAT3 WT refers to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro- flox/ STAT3 MEFs (29), whereas STAT3 KO MEFs refers to mide method were performed as in our previous studies MEFs resulting from deletion of exons 16–21 of STAT3 by (35, 36). infection with a retrovirus encoding Cre recombinase (29). The Hematopoietic Progenitor Cell Assays and Soft Agar Assays— different MEFs were cultured in Dulbecco’s modified Eagle’s Mouse hematopoietic progenitor colony formation was medium supplemented with 10% fetal calf serum and antibiot- assessed as previously described (12, 37). Colony formation ics. A custom-made polyclonal antibody against the N-terminal assays were performed using Sca1 cells isolated from mouse region (amino acids 1–14) of mouse SLFN2 was produced and bone marrow stem cells according to the manufacturer’s purified via New England Peptide LLC (Gardner, MA). Anti- instructions (MACS kit, Miltenyi Biotec Inc., CA). The cells bodies against Cyclin D1, Cyclin D3, CDK 4, CDK 6, p15 INK, were plated in methocult methylcellulose media (Stemcell and p27 KIP were obtained from Cell Signaling Technology Technologies, Seattle, WA) in the presence or absence of 10 (Danvers, MA). An antibody against glyceraldehyde-3-phos- IU/ml IFN, and colony formation was assessed after 7 days of phate dehydrogenase (GAPDH) was obtained from Chemicon culture. Anchorage-independent growth was assessed in soft International (Temecula, CA). An antibody against Lamin A was agar assays in duplicate, carried out essentially as previously purchased from Santa Cruz Biotechnology (Santa Cruz, CA). described (38). Briefly, the cells were suspended in 0.3% top Cell Lysis, Isolation of Nuclear and Cytosolic Fractions, agar over a bottom layer of 0.5% agar in 6-well plates. The solid- Immunoprecipitations, and Immunoblotting—The cells were ified soft agar was overlaid with Dulbecco’s modified Eagle’s lysed in phosphorylation lysis buffer as described in our previ- medium containing 10% fetal bovine serum and antibiotics. ous studies (12, 30). For the detection of IFN-dependent SLFN2 The medium was changed every 4–5 days. The colonies were translocation, the cells were treated with 10 IU/ml IFN for scored after 11 days (NIH3T3 cells) or 8 days (L929 cells) of the indicated times or were left untreated. Nuclear and cytoso- culture. lic fractions were isolated using the Pierce NE-PER kit accord- mRNA Isolation and Real Time PCR Probes and Primers— ing to the manufacturer’s instructions (Thermo Fisher Scien- Cells were treated with 5 10 IU/ml of IFN for the indicated tific, Waltham, MA). For the detection of SLFN2 protein times. Isolation, purification of mRNA, and conversion into expression, cells were treated with 1.5 10 IU/ml IFN for the cDNA was performed using the respective kits and oligo(dT)s 25052 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 1. IFN-inducible expression of SLFN family members. A–E, NIH3T3 cells were treated with IFN for3or6hor left untreated as indicated. Total RNA was subsequently isolated, and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5(D), and SLFN8 (E) was analyzed by real time RT-PCR, using specific primers and GAPDH as an internal control. The data are expressed as fold increase over control untreated samples and represent the means  S.E. of several experiments for SLFN1 (n 3), SLFN2 (n 7), SLFN3 (n 6), SLFN5 (n 4), and SLFN8 (n 5). F, NIH3T3 cells were treated with IFN for 48 h, and after cell lysis, the proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN2 antibody. G, NIH3T3 cells were treated with mouse IFN, as indicated. Cytosolic and nuclear fractions were obtained, and the proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN2 antibody. Immunoblot- ting with antibodies against Lamin A and GAPDH was also performed to control for successful separation of nuclear and cytosolic fractions. from Qiagen according to the manufacturer’s instructions. Val- induction of mRNA expression for key members of the SLFN idated, inventoried probes and primers for real time PCR and gene family was determined. As shown in Fig. 1, mRNA expres- TaqMan PCR master mix were purchased from Applied Bio- sion for different SLFN genes was inducible at various degrees systems (Foster City, CA). The probes and primers were: in response to IFN treatment. The most pronounced induc- SLFN1, Mm00488306_m1; SLFN2, Mm 00488307_m1; SLFN3, tion was for SLFN1 (Fig. 1A), followed by SLFN5, SLFN2, and Mm00488309_g1; SLFN5, Mm00806095_m1; SLFN8, SLFN8 (Fig. 1, B, D, and E). SLFN3 was induced clearly to a Mm00824405_m1; and ISG15, Mm01705338_s1. GAPDH lesser degree than other SLFNs(4-fold), but its induction was (Mm99999915_g1) was used as an internal control. consistently seen (Fig. 1C). To better understand the regulation of SLFN proteins during RESULTS engagement of the Type I IFN receptor, we generated and used In initial studies we determined whether treatment of cells an anti-SLFN2 antibody to directly examine the expression of with IFN induces expression of different SLFN genes. NIH3T3 SLFN2 protein after IFN treatment of cells. This antibody was cells were treated with mouse IFN for different times, and the custom-generated via a commercial vendor against a conserved SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25053 Role of SLFN2 in the Generation of IFN Responses / / FIGURE 2. Differential requirement for STAT1 in IFN-inducible expression of distinct SLFN mRNAs. STAT1 and STAT1 MEFs were treated with IFN for the indicated times. Total RNA was isolated and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5 (D), and SLFN8 (E) mRNAs was determined by real time RT-PCR, after normalization for GAPDH expression. The data are expressed as fold increases over control untreated samples and represent the means  S.E. of two independent experiments for SLFN3 and SLFN5, three for SLFN8, four for SLFN1, and seven for SLFN2. 25054 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 3. Differential requirement for STAT3 in IFN-dependent expression of distinct SLFN mRNAs. The indicated MEFs were treated with IFN for the indicated times. Total RNA was isolated and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5 (D), and SLFN8 (E) mRNAs was determined by real time RT-PCR, after normalization for GAPDH expression. The data are expressed as fold increases over control untreated samples and represent the means S.E. of three independent experiments for SLFN1, SLFN2, and SLFN5 and six for SLFN8. region in the N terminus of the protein and detects a single cells with IFN resulted in up-regulation of the expression of band at44 kDa, which is consistent with the predicted molec- the protein (Fig. 1F). We also examined the subcellular local- ular mass of SLFN2. As shown in Fig. 1F, base-line expression of ization of the protein. In a previous study, it was shown that SLFN2 in NIH3T3 cells was clearly detectable, but treatment of overexpressed FLAG-tagged SLFN2 in HEK-293T cells is SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25055 Role of SLFN2 in the Generation of IFN Responses / / FIGURE 4. Role of p38 MAPK in the regulation of expression of SLFN genes. p38 and p38 MEFs were treated with IFN for the indicated times. Total RNA was isolated and the expression of SLFN1 (A), SLFN2 (B), SLFN3 (C), SLFN5 (D), and SLFN8 (E) mRNAs was determined by real time RT-PCR, after normalization for GAPDH expression. The data are expressed as fold increases over control untreated samples and represent the means  S.E. of three independent experiments for SLFN2, four for SLFN1 and SLFN3, and five for SLFN5 and SLFN8. 25056 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 5. SLFN2 controls hematopoietic progenitor colony formation and promotes the growth-suppressive effects of IFNs on primitive hematopoi- etic precursors. A, to test whether the available siRNAs were efficient and selective for the knockdown of SLFN2 in murine cells, NIH3T3 cells were transfected with control siRNAs or siRNAs selectively targeting SLFN2, and expression of SLFN2 or SLFN3 mRNAs was subsequently examined by real time RT-PCR. Two different pools of SLFN2 siRNA (siRNA1 and siRNA2) and control siRNA (Ctrl siRNA1 and Ctrl siRNA2) were used. The data are presented as percentages of expression in control siRNA transfected cells and represent the means  S.E. of three experiments. B, Sca1 derived, murine hematopoietic progenitor cells were transfected with control siRNA or SLFN2-siRNA, and hematopoietic colony progenitor was assessed in clonogenic assays in methylcellulose. Represent- ative plates are shown. C–D. Sca1 stem cells were isolated from murine bone marrows and plated in methylcellulose in the presence or absence of IFN (C and D). The cells were either not transfected or were transfected with the control nontargeting siRNAs or SLFN2-targeting siRNAs shown in A. Colony formation (colony forming units) of primitive hematopoietic precursors was assessed at day 7 of culture. The data are expressed as percentages of control untransfected cells colony formation and represent means S.E. of five (C) or four (D) independent experiments. Paired t test analysis demonstrated a p value of 0.0004 for IFN-treated Ctrl siRNA1 versus SLFN2 siRNA1 transfected cells (C) and a p value of 0.007 for IFN-treated Ctrl siRNA2 versus SLFN2 siRNA2 transfected cells (D). exclusively expressed in the cytoplasm (25). However, FLAG the effect ranged from a partial impairment (SLFN3) to com- tagging could theoretically interfere with the structural proper- pletely defective transcription (SLFN1, 2, 5, and 8) (Fig. 2). Sim- ties and localization of the protein, and the potential transloca- ilarly, IFN-inducible SLFN expression was examined in tion of endogenous SLFN2 in response to cytokine treatment STAT3 knock-out MEF cells. The induction of expression of has not been known. In studies in which the localization of the SLFN1, SLFN2, SLFN3, and SLFN8 genes was decreased in endogenous protein was directly determined using the newly STAT3 knock-out cells although not abrogated (Fig. 3, A–C). generated anti-SLFN2 antibody, we found that endogenous The expression of SLFN5 was completely STAT3-independent, SLFN2 is exclusively expressed in the cytoplasm, and IFN and in fact, SLFN5 expression was enhanced in STAT3 knock- treatment does not induce its translocation to the nucleus out cells (Fig. 3D). (Fig. 1G). p38 MAPK-activated signaling cascades play important roles IFN binding to the type I IFN receptor results in activation in Type I IFN-dependent transcriptional regulation, acting as of STAT1, STAT2, and STAT3 transcription factors, which auxiliaries to STAT pathways, and their function is essential for form various homo- and/or heterodimers that can bind specific full transcriptional activation of ISGs (reviewed in Refs. 7 and sequences in the promoters of IFN inducible genes (2–7). In 39). To determine the role of p38 MAPK-mediated signals in addition, IFN-mediated gene transcription is regulated by SLFN gene expression, we used MEF cells with targeted disrup- auxiliary pathways, such as the p38 MAPK pathway (7–13). To tion of the p38 gene (27) in which we have previously shown define the roles of distinct STAT proteins and the p38 MAPK in that IFN-inducible transcription via ISRE or GAS elements is SLFN gene expression, experiments were performed using cells defective (33). IFN-dependent mRNA expression for SLFN1, with targeted disruption of STAT1, STAT3,orp38 genes. In SLFN2, and, to a lesser degree, SLFN3 was suppressed in the / / initial studies, STAT1 MEFs and STAT1 parental MEFs absence of p38 MAPK (Fig. 4, A–C). On the other hand, the were treated with mouse IFN, and mRNA expression for group III schlafen genes, SLFN5 and SLFN8, were induced by SLFN1, SLFN2, SLFN3, SLFN5, and SLFN8 was determined. IFN in a p38 MAPK-independent manner (Fig. 4, D and E), IFN-dependent expression of all SLFN genes was decreased in suggesting that p38 activity is essential for IFN-dependent STAT1 knock-out MEFs compared with parental MEFs, and expression of group I and II but not group III Schlafen genes. SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25057 Role of SLFN2 in the Generation of IFN Responses FIGURE 6. Stable knockdown of SLFN2 enhances cell proliferation and impairs IFN-dependent growth inhibitory responses but has no effects on the generation of antiviral responses. A, expression of SLFN2 or SLFN1 mRNAs in pSIREN Zsgreen-SLFN2 siRNA and pSIREN Zsgreen control-siRNA NIH3T3 cells was determined by real time RT-PCR using specific primers and GAPDH as an internal control. The data are presented as percentages of expression in pSIREN Zsgreen control-siRNA cells and represent the means  S.E. of three experiments. B, total cell lysates from pSIREN Zsgreen SLFN2-siRNA or pSIREN Zsgreen control-siRNA NIH3T3 cells were resolved by SDS-PAGE and immunoblotted sequentially with anti-SLFN2 or anti-GAPDH antibodies. C, equal numbers of pSIREN Zsgreen SLFN2-siRNA or pSIREN Zsgreen control-siRNA NIH3T3 cells were plated and were left untreated or were treated with the indicated doses of mouse IFN. After 5–7 days cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays. A representative experiment is shown in the left panel. The means S.E. of three experiments, including the one shown in the picture on the left, are shown on the right panel. D and E, pSIREN Zsgreen SLFN2 siRNA or pSIREN Zsgreen control-siRNA NIH3T3 cells were treated with IFN for 15 min, as indicated. Nuclear extracts were reacted with 40,000 cpm of P-labeled ISRE (D) or SIE (E) oligonucleotides, and complexes were resolved by native gel electrophoresis and visualized by autoradiography. The migration of the different STAT complexes is indicated by arrows. F, wild-type NIH3T3 cells, pSIREN Zsgreen SLFN2 siRNA NIH3T3 cells, or pSIREN Zsgreen control-siRNA NIH3T3 cells were treated with IFN for the indicated times. Expression of Isg15 mRNA was determined by real time RT-PCR using GAPDH as an internal control. The data are expressed as the means S.E. of three experiments. G, wild-type NIH3T3 cells, pSIREN Zsgreen SLFN2-siRNA NIH3T3 cells, or pSIREN Zsgreen control-siRNA NIH3T3 cells were incubated in triplicate, in the presence or absence of the indicated concentrations of IFN. The cells were subsequently challenged with encephalomyocarditis virus (EMCV), and cytopathic effects were quantified 24 h later. The data are expressed as percent- ages of protection from the cytopathic effects of encephalomyocarditis virus. A representative of three independent experiments is shown. It is well known that Type I and II IFNs are potent regulators as expected, treatment of cells with IFN (Fig. 5, C and D)or of normal and leukemic hematopoiesis and inhibit the growth IFN (data not shown) resulted in suppression of hematopoi- of primitive hematopoietic precursors in vitro and in vivo (4, 39, etic progenitor colony formation compared with untreated 40). It has been also established that activation of the p38 MAP cells, although the suppressive effects of IFN were much less kinase pathway is required for the generation of the myelosup- noticeable in cells in which SLFN2 was knocked down (Fig. 5, C pressive effects of IFNs on both normal and leukemic progeni- and D). Thus, SLFN2 participates in the control of normal tors (12, 13). Because SLFN2 is induced by IFNs and its expres- hematopoiesis and the generation of the myelossuppressive sion is regulated via both STAT and p38 MAPK pathways, we effects of IFNs, suggesting that this protein may be an effector examined whether this protein plays a role in the generation of in the regulation of p38-mediated hematopoietic suppression. the myelosuppressive effects of IFN. In initial experiments, To further analyze the functional relevance of SLFN2 in cell two different specific siRNAs (Fig. 5A) were used to knock growth regulation and its role in the generation of IFN down SLFN2 expression in murine bone marrow-derived responses in other cell types, we generated stable SLFN2 knock- Sca1 stem cells. Primitive progenitor colony formation was down NIH3T3 cells via expression of shRNA-targeting SLFN2 subsequently assessed in clonogenic assays in methylcellulose. using the pSIREN Zsgreen retroviral system. SLFN2 expression Knockdown of SLFN2 in normal hematopoietic progenitors was selectively knocked down in NIH3T3 cells (Fig. 6, A and B). resulted in increased hematopoietic colony formation (Fig. 5, Cells in which SLFN2 was knocked down exhibited enhanced B–D), suggesting that this protein plays a critical role in the proliferation compared with their control counterparts (Fig. control of normal hematopoietic progenitor cell growth. Also, 6C). IFN treatment resulted in dose-dependent growth sup- 25058 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 6 —continued pression in both NIH3T3 pSIREN Zsgreen Ctrl siRNA and appears to be impairing IFN-dependent cell cycle arrest but NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells (Fig. 6C). How- not IFN-inducible gene transcription or generation of antiviral ever, in NIH3T3 cells in which SLFN2 was knocked down, responses. IFN-induced antiproliferative responses were clearly To examine whether SLFN2 plays a role in the control of decreased compared with cells expressing SLFN2 (Fig. 6C), anchorage-independent growth, we assayed transduced indicating that SLFN2 participates in the generation of the NIH3T3 cells for colony formation in soft agar (41). Colony growth inhibitory effects of IFN. On the other hand, SLFN2 formation was clearly increased in NIH3T3 pSIREN Zsgreen knockdown had no effect on IFN-dependent formation of SLFN2-siRNA cells as compared with NIH3T3 pSIREN STAT-containing DNA-binding complexes (Fig. 6, D and E). Zsgreen Ctrl siRNA cells (Fig. 7, A and B). Notably, the colonies Similarly, IFN-dependent Isg15 gene transcription (Fig. 6F)or from NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells were con- generation of IFN-induced antiviral responses (Fig. 6G) were sistently larger (Fig. 7A), and the numbers of colonies were not affected by SLFN2 knockdown. Thus, targeting SLFN2 increased (Fig. 7B) as compared with NIH3T3 pSIREN Zsgreen SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25059 Role of SLFN2 in the Generation of IFN Responses FIGURE 7. Effects of SLFN2 knockdown on anchorage-independent growth. A, equal numbers of NIH3T3 pSIREN Zsgreen control-siRNA and NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells were plated in a soft agar assay system. Colony formation was analyzed after 11 days of culture. Representative areas of the soft agar plates for NIH3T3 pSIREN Zsgreen control-siRNA and NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells are shown. B, colonies were counted, and the results were expressed as percentages of control of NIH3T3 pSIREN Zsgreen control-siRNA-derived colonies. The data shown represent the means  S.E. of three inde- pendent experiments, including the one shown in A. Paired t test analysis showed a p value of 0.01. although additional mechanisms may be involved, these findings sug- gest that SLFN2 inhibits cell growth and colony formation in part via suppression of cyclin D1 and up-regulation of the CDK inhibitor p15 INK. To definitively establish the role of SLFN2 in the generation of IFN responses and anchorage-indepen- FIGURE 8. SLFN2 knockdown in NIH3T3 cells modulates expression of Cyclin D1 and p15 INK cell cycle dent growth in nonhematopoietic regulators. NIH3T3 pSIREN Zsgreen control-siRNA and NIH3T3 pSIREN Zsgreen SLFN2-siRNA cells were syn- cells, we stably knocked down chronized via serum starvation. After re-entry of cells into the cell cycle by the addition of serum, the cells were collected at the indicated time points. Total cell lysates were resolved by SDS-PAGE and immunoblotted with SLFN2 in another murine fibroblast antibodies against Cyclin D1 (A) or p15 INK (B), as indicated. cell line, L929. Initially, we exam- ined the IFN-inducible expression Ctrl siRNA cells. Taken altogether, these data for the first time of SLFN2 in L929 cells. The cells were treated with mouse IFN implicate SLFN2 in the regulation of anchorage-independent for different times, and the induction of mRNA and protein growth. expression was analyzed. As expected, both SLFN2 mRNA (Fig. In subsequent studies, we sought to obtain information on 9A) and protein (Fig. 9B) expression were up-regulated in the mechanisms by which SLFN2 regulates anchorage-inde- response to IFN treatment. We then generated stable SLFN2 pendent cell growth and blocks cell proliferation. Initially, we knockdown L929 cells via expression of shRNA-targeting examined the effects of SLFN2 knockdown on the expression of SLFN2 using the same pSIREN Zsgreen retroviral system we various key cell cycle regulators. We compared the levels of utilized before to knock down SLFN2 in NIH3T3 cells. Green expression of Cyclin D1, Cyclin D3, CDK4, CDK6, and the CDK fluorescent L929 pSIREN Zsgreen cells were selected after ret- inhibitors p27 KIP1 and p15 INK in serum-starved and cycling roviral transfection and analyzed for SLFN2 expression. As NIH3T3 control cells or NIH3T3 cells in which SLFN2 was shown in Fig. 9 (C and D), stable SLFN2 expression was selec- knocked down. As shown in Fig. 8A, stable SLFN2 knockdown tively knocked down in L929 pSIREN Zsgreen SLFN2-siRNA in NIH3T3 cells resulted in higher basal Cyclin D1 levels of cells compared with L929 pSIREN Zsgreen Ctrl siRNA cells. expression than control cells, whereas Cyclin D1 levels were We next analyzed IFN-dependent Isg15 gene transcription also consistently higher in cycling SLFN2 knockdown cells (Fig. 9E) in SLFN2 stable knockdown L929 cells, as well as the compared with control cells (Fig. 8A). On the other hand, effects of stable SLFN2 knockdown on IFN-induced antipro- Cyclin D3, as well as CDK4 and CDK6, levels were not signifi- liferative responses (Fig. 9F). Consistent with the results cantly altered in cells in which SLFN2 was knocked down across obtained with NIH3T3 cells, L929 cells with stable SLFN2 the time points analyzed (data not shown). When the levels of knockdown showed enhanced proliferation and were less sen- expression of the CDK inhibitors p27 KIP1 and p15 INK were sitive to the suppressive effects of IFN compared with their assessed, we noticed that unlike p27 expression, which was not control counterparts (Fig. 9F), whereas IFN-dependent Isg15 consistently altered (data not shown), p15 INK levels were gene transcription was unaltered (Fig. 9E). clearly lower in resting and cycling NIH3T3 pSIREN Zsgreen SLFN2-siRNA transfected cells, compared with NIH3T3 pSI- We also determined whether SLFN2 knockdown in L929 REN Zsgreen Ctrl siRNA transfected cells (Fig. 8B). Thus, cells enhances anchorage-independent growth. L929 pSI- 25060 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses FIGURE 9. Stable knockdown of SLFN2 enhances cell proliferation and impairs IFN-dependent growth inhibitory responses. A, L929 cells were treated with IFN for3or6hor left untreated as indicated. Total RNA was subsequently isolated, and the expression of SLFN2 mRNA was analyzed by real time RT-PCR, using specific primers for SLFN2 and GAPDH as an internal control. The data are expressed as fold increases over untreated samples and represent the means S.E. of four independent experiments. B, L929 cells were either left untreated or were treated with IFN for 24 or 48 h, as indicated. After cell lysis, the proteins were resolved by SDS-PAGE and immunoblotted with anti-SLFN2 or anti-GAPDH antibodies, as indicated. C, expression of SLFN2 or SLFN3 mRNAs in L929 Zsgreen-Ctrl siRNA and pSIREN Zsgreen SLFN2-siRNA in L929 cells was determined by real time RT-PCR using specific primers and GAPDH as an internal control. The data are presented as percentages of expression in pSIREN Zsgreen control-siRNA cells and represent means S.E. of five experiments. D, total cell lysates from pSIREN Zsgreen SLFN2-siRNA or pSIREN Zsgreen control-siRNA L929 cells were resolved by SDS-PAGE and immunoblotted sequentially with anti-SLFN2 or anti-GAPDH antibodies. E, wild-type L929 cells, pSIREN Zsgreen SLFN2 siRNA L929 cells or pSIREN Zsgreen control-siRNA L929 cells were treated with IFN for the indicated times. Expression of Isg15 mRNA was determined by real time RT-PCR using GAPDH as an internal control. The data are expressed as fold increases over control untreated cells and represent the means  S.E. of four experiments. F, equal numbers of L929-pSIREN Zsgreen SLFN2-siRNA or L929-pSIREN Zsgreen control-siRNA cells were either left untreated or treated with the indicated doses of mouse IFN for 5 days, and cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays. The means  S.E. of three experi- ments are shown. SEPTEMBER 11, 2009• VOLUME 284 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 25061 Role of SLFN2 in the Generation of IFN Responses FIGURE 10. Effects of SLFN2 knockdown on anchorage-independent growth of L929 cells. A, equal numbers of L929 pSIREN Zsgreen control-siRNA and L929 pSIREN Zsgreen SLFN2-siRNA cells were plated in a soft agar assay system. Colony formation was analyzed after 8 days of culture. Representative areas of the soft agar plates for L929 pSIREN Zsgreen control-siRNA and L929 pSIREN Zsgreen SLFN2-siRNA cells are shown. B, colonies were counted, and the results were expressed as percentages of control of L929 pSIREN Zsgreen control-siRNA-derived colonies. The data shown represent the means  S.E. of four independent experiments, including the one shown in A. Paired t test analysis showed a p value of 0.04. REN Zsgreen SLFN2-siRNA and L929 pSIREN Zsgreen Ctrl Superfamily I DNA/RNA helicase motif not found in group I/II siRNA cells were plated, and colony formation was deter- SLFNs, whereas the members of this group are significantly mined after 8 days of culture in soft agar. As depicted in Fig. larger proteins with molecular masses ranging between 100 10, L929 cells with stable SLFN2 knockdown showed consis- (SLFN5) and 104 kDa (SLFN8) (21). Although the roles of tently larger colonies (Fig. 10A), and there were increased members of this SLFN group remains to be established, studies numbers of colonies compared with L929 pSIREN Zsgreen with SLFN8 transgenic mice have suggested an important reg- Ctrl siRNA cells (Fig. 10B). ulatory role for this SLFN gene in T cell development and dif- ferentiation (21). Notably, different SLFN family members have DISCUSSION been shown to be induced in response to a wide variety of stim- The family of Schlafen genes was originally identified during uli, including CpG-DNA (24), the bacterial pathogens Brucella screening for growth regulatory genes that are differentially and Listeria (44), and terminal differentiation of myeloid cells expressed during lymphocyte development (20). Originally, (21), suggesting that signals from divergent stimuli converge on SLFN family members 1, 2, 3, and 4 were identified and studied SLFN family members to control cell cycle progression. (20). Initial studies had suggested that SLFN genes suppress Despite the fact that studies on the functional relevance and growth and participate in the maintenance of the quiescent biochemical activities of SLFN proteins have been very limited state of naive T lymphocytes, as shown by experiments involv- so far, the emerging evidence suggests key regulatory roles for ing ectopic expression of SLFN1, demonstrating disruption of these proteins on cell cycle progression and growth arrest. Yet thymic development (20). Subsequently, and based on very little is known on their potential involvement in the gen- sequence homology, Geserick et al. (21) identified additional eration of the suppressive effects of growth inhibitory cyto- SLFN genes (SLFN5, SLFN8, SLFN9, and SLFN10) forming a kines. Type I IFNs are probably the most prominent cytokines cluster on mouse chromosome 11 where the SLFN1–4 genes that generate growth inhibitory and antitumor effects; and are also located. these properties have over the years led to their introduction The different members of the SLFN family of proteins can be in the treatment of various leukemias and solid tumors (3). Impor- classified into three subgroups (20, 21). The first group includes tantly, although it is well established that IFNs regulate cell cycle SLFN1 and SLFN2, which encode for the smallest two SLFN progression and induce G /G cell cycle arrest, very little is 0 1 proteins, with predicted molecular masses of 37 and 42 kDa, known about the IFN-inducible proteins that mediate such respectively (20). They contain an AAA domain, found in responses. In the present study, we provide the first evidence ATPases (42), and an adjacent “SLFN box,” which is common to that IFNs regulate expression of members of the SLFN family of all SLFN proteins (21, 25). Overexpression of SLFN1 results in genes and proteins. Our data demonstrate that IFN is a potent potent growth suppression by inducing G cell cycle arrest (20) inducer of different SLFN family members, including members through inhibition of cyclin D1 expression (22). In addition, it of Group I (SLFN1 and SLFN2), Group II (SLFN3), and Group appears that accumulation of SLFN1 protein to the nucleus III (SLFN5 and SLFN8). Moreover, in work aimed to define the correlates with induction of its growth-suppressive effects (43). regulation of expression of these proteins by IFNs, we estab- The second group of SLFN proteins includes SLFN3 and lished the differential involvement of distinct IFN-activated SLFN4, which have predicted molecular masses of 58 and 68 kDa, respectively. These proteins have in their structures a STAT proteins and the p38 MAP kinase in their regulation. small sequence motif (SWA(L/V)DL) (21, 25), also shared by Our finding that members of the SLFN family of proteins are the third group. This third group of SLFN proteins contains a engaged by the Type I IFN receptor in a STAT- and/or p38 25062 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 37 •SEPTEMBER 11, 2009 Role of SLFN2 in the Generation of IFN Responses MAPK-dependent manner provided a direct link between IFN- development of methodologies to selectively induce SLFN gene activated Jak-STAT pathways and cellular elements controlling expression may specifically promote the antitumor effects of cell cycle progression. Such a link led us to further studies IFNs in the absence of engagement of other pathways associ- aimed to define the functional relevance of the SLFN pathway ated with various IFN-inducible adverse effects. Although the in the generation of IFN responses. 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Published: Sep 1, 2009

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