Oncogene (2005) 24, 5278–5286 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc

The Runx1 controls CSF-1-dependent and -independent growth and survival of macrophages

Stewart R Himes1, Stephen Cronau1, Christine Mulford1 and David A Hume*,1

1CRC for Chronic Inflammatory Disease, Institute for Molecular Biosciences, Queensland Biosciences Precinct, Bldg. 80, Services Rd., University of Queensland, Brisbane, Queensland 4072, Australia

Gene translocations that repress the function of the Runx1 (Tracey and Speck, 2000). Runx1 has been shown to transcription factor play a critical role in the development be involved in expression of numerous tissue-specific of myeloid leukemia. In this report, we demonstrate that and can cooperatively activate transcription in Runx1 precisely regulates c-fms (CSF-1 receptor) concert with tissue-specific transcription factors such as expression. Runx1 controlled expression by binding to C/EBPa, PU.1 and c-Myb (Zhang et al., 1996a, b; multiple sites within the mouse c-fms gene, allowing Lutterbach and Hiebert, 2000). The importance of interaction between promoter and downstream enhancer Runx1 and CBFb in normal and malignant hematopoi- elements. The and c-fms genes showed an identical esis is evident from frequent translocations in both genes pattern of expression in mature macrophages. Runx1 in leukemia and a complete loss of fetal hematopoiesis expression was repressed in CSF-1 stimulated, proliferat- by targeted disruption of either gene (Miyoshi et al., ing bone marrow-derived macrophages (BMM) and 1993; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., significantly increased in quiescent, CSF-1 starved cells. 1996). The RAW264.7 and Mono-Mac-6, macrophage-like cell Translocations in CBF genes result in fusion lines expressed low levels of Runx1 and both showed that act as dominant inhibitors of native CBF function. growth arrest and cell death with ectopic expression of The t(8;21)translocation fuses nearly all of the Runx1. The EM-3 cell line, which represents an early transcriptional repressor ETO to the N-terminus of myeloid progenitor cell line, showed growth arrest with Runx1, resulting in replacement of the C-terminal trans- Runx1 expression in the absence of any detectable activation domain of Runx1 with the histone deacety- changes in cell differentiation. These findings suggest that lase domain of ETO (Runx1-ETO)(Downing et al., Runx1 regulates growth and survival of myeloid cells and 1993; Meyers et al., 1995). The inv(16) translocation provide a novel insight into the role of Runx family gene fuses MYH11, a smooth muscle myosin heavy chain translocations in leukemogenesis. gene, to CBFb(CBFb-SMMC). The resulting fusion Oncogene (2005) 24, 5278–5286. doi:10.1038/sj.onc.1208657; interferes with nuclear localization of the CBF published online 27 June 2005 protein complex (Thompson et al., 1991; Adya et al., 1998). Ectopic expression in transgenic mice showed Keywords: Runx1; macrophage; CSF-1; survival that although these fusion proteins can disrupt normal hematopoiesis, they are not sufficient for the develop- ment of leukemia (Castilla et al., 1999; Heibert et al., 2001). The link between loss of CBF function and leukemia Introduction suggests that CBF proteins act as strong tumor suppressors. Many genes that reside at breakpoint The AML-1/Runx1 transcription factor is a common regions in leukemogenic translocations and numerous target of gene translocation in leukemia (Maruyama tumor suppressor genes are important regulators of cell et al., 1993; Heibert et al., 2001). The DNA-binding proliferation and survival. Loss of Runx1 function was activity of this protein was originally characterized as believed to impact primarily on cell differentiation core binding factor (CBF)based upon the ability to bind resulting in abnormal accumulation of progenitor cells. the core enhancer sequence of the Moloney Leukemia A recent report used Cre/Lox inducible disruption of Virus LTR. CBF was subsequently found to comprise a exon 5 to show that myeloid differentiation in adult complex of Runx1 and a non-DNA-binding subunit, mice was not affected by loss of Runx1 expression CBFb (Wang and Speck, 1992). Runx1 and CBFb are (Ichikawa et al., 2004). Normal myeloid development in normally expressed in all hematopoietic tissues, during the absence of Runx1 expression is not consistent with myeloid differentiation and in mature macrophages lineage progression being the primary role of Runx1 in suppression of myeloid leukemia. *Correspondence: DA Hume; E-mail: [email protected] Several studies have indicated that Runx1 activity Received 19 October 2004; revised 18 January 2005; accepted 3 February may extend beyond regulation of cell differentiation. 2005; published online 27 June 2005 Runx1-ETO expression in mice resulted in the presence Runx1 regulation of macrophage growth and survival SR Himes et al 5279 of hematopoietic progenitors that showed aberrant from a highly conserved intronic enhancer, termed the proliferation in vitro with an increased capacity for Fms intronic regulatory element (FIRE)(Himes et al., self-renewal (Okuda et al., 1998). Similar results were 2001), which has additional potential Runx1 binding obtained from ectopic expression of Runx1-ETO in sites (Tagoh et al., 2002; Follows et al., 2003). Since hematopoietic stem and progenitor cells using retroviral CSF-1R is similarly expressed in human and mouse transduction. Runx1-ETO expression caused a strong macrophages, we anticipated that the key control enhancement in self-renewal of hematopoietic stem cells mechanisms would also be conserved across species. but inhibited colony formation by committed progeni- We therefore carried out experiments to determine tors in response to cytokine treatment (Mulloy et al., whether Runx1 regulates expression of the mouse gene 2002). and to determine whether activation involves putative Part of the reason why studies of Runx1-associated conserved binding sites in the FIRE enahncer. Towards leukemogenesis have focused on its role in differentia- this end, we produced expression constructs for both tion is that the protein is a transcriptional regulator of full-length Runx1 (PEBP2aB)and CBF b proteins based known differentiation associated genes. For example, on the pEF6 expression plasmid that uses the EF-1a Runx1 may control macrophage development by promoter for stable expression of genes. Stable transfec- regulating CSF-1 receptor (CSF-1R)expression (Zhang tion of the macrophage-like RAW264.7 cell line with et al., 1996a). CSF-1 (also termed macrophage-CSF) is either the pEFRunx1 or pEFCBFb expression con- required for macrophage development as determined by structs was performed and cells were analysed for CSF-1R the phenotype of mice with a targeted deletion of the expression by antibody staining and flow cytometry. CSF-1R or a natural mutation in the CSF-1 gene The level of CSF-1R expression in RAW264.7 cells was (Cecchini et al., 1997; Dai et al., 2002). Previous work low, but increased significantly with expression of either showed that Runx1 can synergistically activate the Runx1 or CBFb. Runx1 expression alone resulted in a human c-fms (CSF-1R)gene promoter with PU.1 and greater than 10-fold increase in surface expression of C/EBP proteins (Zhang et al., 1996a; Petrovick et al., CSF-1R when compared to the empty vector (pEF6) 1998). The binding site in the human c-fms promoter, control (Figure 1). Expression of the CBFb cofactor also however, is not conserved between human and mouse. enhanced CSF-1R expression but to a lesser extent than In this report, we characterize multiple binding sites Runx1. This finding represents the first direct evidence within the human and mouse genes and demonstrate that expression of the CSF-1R gene product, as opposed that Runx1 is also an important regulator of the mouse to promoter activity, can be controlled by Runx1. c-fms gene. c-fms Although is a differentiation Mouse and human c-fms genes contain multiple CBF et al marker for the macrophage lineage (Tagoh ., 2002; binding sites Sasmono et al., 2003), signaling from CSF-1 receptor encoded by this gene is also involved in macrophage Sequence analysis of regulatory regions revealed that the survival and proliferation. Removal of CSF-1 from location of CBF binding sites within the mouse and macrophage cultures leads to cessation of proliferation, human c-fms genes may not be conserved (Himes et al., and eventually to programmed cell death. An early 2001). Electrophoretic mobility shift assays (EMSA) event in this pathway is rapid upregulation of the CSF-1 were used to identify binding sites in either the promoter receptor. We report here that expression of the Runx1 or enhancer of the mouse and human genes. Specific transcription factor is itself acutely regulated by CSF-1 antibody to the Runx1 protein was used to identify and may contribute to both CSF-1 receptor upregula- Runx1 containing protein complexes by antibody super- tion and the control of cell survival and proliferation at shift. This analysis showed that both the human and later stages of macrophage development. We further mouse genes contain two sites that bind CBF proteins show that ectopic Runx1 expression resulted in growth with high affinity. In the mouse gene, both CBF binding arrest of macrophage-like and early myeloid progenitor sites were present in the enhancer. There was an cell lines. Our data suggest a dual role for Runx1 as an upstream site whose sequence showed no conservation important factor for macrophage development through with the human gene enhancer by alignment (MRxE) CSF-1R expression and as an inhibitor of cell prolifera- and a second downstream element which was completely tion or survival by restricting cytokine-independent conserved (HMRxE)(Figure 2a).The sequence within proliferation. the mouse promoter that corresponds to the previously characterized human element contains a single base change from the TGTGGT core consensus and showed weak CBF protein binding (MRxP). In the human gene, Results the sites are separated, one in the promoter and the Runx1 activates expression of the mouse CSF-1R gene conserved site in the enhancer (HRxP and HMRxE). All sites bound multiple protein complexes, which can be Runx1 has been shown to trans-activate the human supershifted with Runx1 antibody (Figure 2b). c-fms gene proximal promoter, but the core consensus The relative affinity of Runx1 binding sites was Runx1 binding site is not conserved in the mouse c-fms determined by ability to compete for CBF binding to a gene promoter. Furthermore, the appropriate expres- consensus site in the Molony Leukemia Virus LTR, sion of the c-fms gene actually requires a contribution PEBP2a (Satake et al., 1992). Unlabeled oligonucleotide

Oncogene Runx1 regulation of macrophage growth and survival SR Himes et al 5280 complete competition for CBF binding with the PEBP2a probe. The conserved site in the enhancer that shows a T to G substitution (HMRxE)in the final base of the core consensus sequence was also able to compete for CBF binding, but was less effective compared to the consensus elements (Figure 2c).

Runx1 trans-activates the mouse CSF-1R promoter through the intronic enhancer Luciferase reporter constructs were used to determine whether Runx1 can function at the level of transcription through either promoter or enhancer elements of the mouse c-fms gene. We also wished to determine if Runx1 expression would alter the position dependence of FIRE enhancer activity. Our previous work showed that the intronic enhancer of the c-fms gene was highly active when inserted proximal to the c-fms gene promoter and insertion distal to the promoter, downstream of the luciferase coding region, resulted in substantial loss of enhancer activity in RAW264.7 cells (Himes et al., 2001). This speculation was based on the fact that the CBF protein complex is capable of interacting with numerous transcription factors and cofactors, including the PU.1 protein which can bind sites in both the human and mouse promoters (Petrovick et al., 1998). These results have suggested that CBF proteins may act as organizing factors, allowing the recruitment of proteins that bind distal elements into the transcription complex. Consistent with the lack of high-affinity binding sites, CBF proteins were capable of only weak trans-activa- tion of the mouse c-fms gene proximal promoter alone. In RAW264.7 cells, the presence of the enhancer permitted a significant increase in CBF activation whether the enhancer was located proximal (p0.5fmsUE)or distal (p0.5fmsDE)to the promoter. The constitutive activity of the two enhancer constructs varied considerably. The p0.5fmsUE reporter construct showed strong enhancer activity even in the absence of Runx1/CBFb expression resulting in a six fold increase in expression through the c-fms gene promoter. The p0.5fmsDE construct required CBF co-transfection for high-level enhancer activity. In the absence of CBF Figure 1 Runx1 activates cell surface expression of CSF-1 receptor. RAW264.7 cells were transfected with either (a)the expression, the distal enhancer produced a less than pEF6 empty vector control, or the pEFRunx1, pEFCBFb for twofold increase in reporter gene activity. Expression of stable expression of (b)Runx1 or ( c)CBF b. Transfected cells were CBF proteins resulted in a significant increase in stained with a monoclonal antibody specific for the CSF-1 receptor enhancer activity, resulting in an over five-fold increase (a-fms)or an isotype control (I.C.).The Y-axis shows the number in reporter expression over the promoter alone of cells and the X-axis the level of immunofluorescence on a logarithmic scale (Figure 3). The position of the enhancer in p0.5fmsDE, downstream from the start of transcription, more closely mimics positioning within the endogenous CSF-1R gene. Low-level expression in the absence of CBF from each of the c-fms binding sites was added to the protein expression was also consistent with the expres- binding reaction at a ratio of 20 : 1 with the labeled sion pattern of the CSF-1R gene in RAW264.7 cells PEBP2a probe. The results showed that the G to A (Figure 1). substitution in the core consensus sequence (TGTGGT), seen in the mouse c-fms gene promoter, significantly Runx1 expression is induced upon removal of CSF-1 from reduced the affinity for CBF. The consensus elements in macrophages the human c-fms promoter site and the site in the mouse enhancer that is not conserved in the human enhancer Surface CSF-1R, and c-fms mRNA expression is both showed a strong affinity for CBF with nearly relatively low in macrophages exposed to saturating

Oncogene Runx1 regulation of macrophage growth and survival SR Himes et al 5281

Figure 2 EMSA of Runx1 binding sites in the c-fms gene. EMSA analysis was performed on potential Runx1 binding sites within the mouse, M or human, H, c-fms genes. Oligonucleotides containing elements from the gene promoter, P or downstream intronic enhancer, E (e.g. mouse Runx1 site in enhancer, MRxE), were labelled and incubated with nuclear extract from BMM for protein binding. (a)Sequence of the conserved intronic enhancer from mouse and human showing Runx1 binding sites. ( b)Polyacrylamide gel showing Runx1/CBFb protein binding to the c-fms gene elements shown above. Runx1 containing protein complexes are indicated on the gel as identified by the ability of Runx1 specific antibody ( þ Ab)to supershift the protein complex (Runx1 SS).( b)Competition for Runx1 protein binding with the PEBP2a element in the MMLV LTR. Competitor oligonucleotides were added at a ratio of 20 : 1 as indicated above the gel amounts of CSF-1, and is inducible upon CSF-1 mouse RAW264.7 macrophage line, or in the human removal (Hume et al., 1997). The mechanism of this mono-mac-6 pro-monocytic cell line. feedback loop has not previously been identified. Since Runx1 is a potent trans-activator of the c-fms gene, we Inhibition of cell proliferation by Runx1 analysed the effect of CSF-1 removal on expression of Runx1 mRNA. Bone marrow-derived macrophages The reversible induction of runx1 following growth (BMM), which are dependent upon CSF-1 for prolif- factor deprivation in BMM suggests that runx1 may act eration and survival, were either maintained in CSF-1, as a growth or survival inhibitor in myeloid cells in or depleted of CSF-1 for 16 h, and the level of Runx1 addition to its action on CSF-1R expression. We had mRNA was measured by real-time RT–PCR analysis. previously noted that attempts to achieve stable over- Runx1 expression increased 4.3-fold when BMM were expression of Runx1 in RAW264.7 cells were unsuccess- deprived of CSF-1 for 16 h. Subsequent treatment of ful, and were associated with a substantial reduction in starved BMM with CSF-1 resulted in a rapid decline in the number of primary transformants (colonies)com- Runx1 expression after 4 h of treatment with a greater pared to empty vector controls. To quantitate this than 80% decrease in expression compared to CSF-1 phenomenon, cell viability assays (based upon MTT dye deprived cells (Figure 4a, b). reduction assay; see Materials and methods)were Western blot analysis was used to determine the level performed immediately after antibiotic selection to of Runx1 protein expression in cell lines and proliferat- evaluate the effect of ectopic expression on the growth ing or quiescent BMM. CSF-1-stimulated BMM showed and viability of transfected cell lines. Runx1- expressing a low-level expression of Runx1 protein that increased cells showed a 72% decrease in viable cells and CBFb- significantly when BMM were deprived of CSF-1 expressing cells a 41% decrease compared to nonexpres- (Figure 4c). For comparison, the level of Runx1 sing cells transfected with the empty vector, pEF6. expression in starved BMM was found to be signifi- Since RAW264.7 cells are a mouse line, and cantly higher than in the growth factor-independent transformed with the v-abl oncogene, we wished to

Oncogene Runx1 regulation of macrophage growth and survival SR Himes et al 5282

Figure 3 CBF proteins trans-activate regulatory elements from the mouse c-fms gene. (a)Diagram of luciferase reporter constructs containing the mouse c-fms gene promoter with or without the downstream intronic enhancer. (b)In all, 5 mg of each reporter plasmid was cotransfected into RAW264.7 cells with either 10 mgof the empty vector, pEF6 or 5 mg each of the pEFRunx1 and pEFCBFb expression plasmids. At 32 h post-transfection, protein was extracted from cells and assayed for luciferase activity. Columns on the graph show the mean value and error bars the s.e.m. of three independent replicate transfections. Values were computed relative to the concentration of protein extract as defined in the materials and methods

determine whether growth regulation by Runx1 was more universal across species and cell systems. The ability of Runx1 to control cell growth was also examined in the human promonocytic cell line, Mono- Mac-6. Stable transfection with either Runx1 or CBFb again resulted in strong inhibition of cell growth and viability. Stable expression of Runx1 resulted in a 90% decline in viable cells compared to pEF6-transfected cells. Similar to RAW264.7 cells, expression of CBFb had a significantly lower effect on cell growth (Figure 5). Stable transfection was performed on the BCR-Abl transformed EM-3 cell line that showed expression of Runx1 protein by Western blot (Figure 4). CBFb expression did not result in a significant change in the number of EM-3 cells but expression of Runx1 resulted Figure 4 CSF-1 stimulation inhibits expression of runx1 and c-fms in a 42% decline in viable cells (Figure 5). mRNA. A time course of CSF-1 stimulation was performed on BMM. mRNA was isolated from cells at 4 h intervals after CSF-1 depletion followed by retreatment with 1000 units of CSF-1. The Runx1 acts as a growth inhibitor but not a differentiation level of (a) runx1 mRNA or (b) c-fms mRNA was quantitated using real time PCR. Columns show the mean level of mRNA and the factor in EM-3 cells error bars the s.e.m. of three replicate PCR reactions. Values are Inhibition of cell proliferation or viability by expression given relative to the level of HPRT mRNA, which functions as an internal control. (c)Western blot of nuclear protein to determine of Runx1 could be explained by changes in the the level of Runx1 expression in the Mono-Mac-6 (MM6), EM-3 differentiation state of cells. Although both the and RAW264.7 cell lines and in CSF-1 stimulated ( þ )or deprived RAW264.7 and Mono-Mac-6 cell lines display macro- (À)primary macrophages (BMM).Data are representative of at phage-like properties and are positive for most mono- least three replicate experiments cyte/macrophage cell markers, Runx1-mediated

Oncogene Runx1 regulation of macrophage growth and survival SR Himes et al 5283

Figure 5 Expression of Runx1 inhibits cell growth or viability. The RAW264.7, Mono-Mac-6 and EM-3 cell lines were transfected with either the pEF6, empty vector or the pEFRunx1 and pEFCBFb vectors for stable expression of Runx1 or CBFb.An MTT assay was performed on transfected cells to determine the level of cell viability. Values are normalized by setting the mean absorbance value of the empty vector control as 100. Columns represent the mean value and error bars the s.e.m. of three independent stable transfection pools. *P>0.05 by small sample t-test of the mean repression of growth or survival may still result from terminal differentiation of cells. EM-3 cells expressed Runx1 and showed reduced toxicity with ectopic expression of Runx1 compared to the pro-monocytic cell lines. EM-3 cells appear to be early progenitors and are negative for most myeloid cell surface markers except the CD13 and CD15 myeloid cell surface markers (German Tissue Culture Collection, DMSZ). Unlike RAW264.7 or Mono-Mac-6 cells, ectopic expression in EM-3 cells did not result in significant changes in cell morphology or the ability of cells to adhere to tissue culture plastic (data not shown). The data are also consistent with no change in myeloid development with inducible knockout of Runx1 (Ichikawa et al., 2004). A more detailed analysis of cell cycle and cell death using propidium iodide was performed on transfected EM-3 cells since significant numbers of cells could be obtained after antibiotic selection. EM-3 cells over- expressing Runx1 showed a 9.5-fold increase in the rate of cells undergoing apoptosis, as defined by cells containing less than 2N DNA, and a 36% decrease in Figure 6 Runx1 expression decreases cell proliferation or survival. the number of cells in cycle. In contrast, expression of EM-3 cells that overexpress Runx1 were assayed for the number of CBFb resulted in relatively minor effects on growth or cells in cycle or undergoing apoptosis by propidium iodide staining. survival (Figure 6a, b, c). The level of expression of Graphs show the cell cycle profiles of (a)EM-3 cells transfected either Runx1 or CBFb protein was determined in three with the empty vector pEF6, (b)the pEFRunx1 or ( c)pEFCBFb expression vectors. The percentage of cells at different stages of the independent stable transfection pools by Western blot cell cycle are shown below and correspond to the G0/G1 analysis of V5 epitope tagged protein. The level of checkpoint, for cells with 2N DNA, S for cells in DNA synthesis protein expression was similar indicating that changes in and the G2 checkpoint for cells with 4N DNA. Cells with less than growth and survival were not due to significant 2N DNA undergoing apoptosis are designated as A. Data are representative of three independent stable transfection pools. differences in the level of either Runx1 or CBFb protein (d)Western blot analysis for Runx1 or CBF b protein expression expression. The level of expression of the developmental in the three stable transfection pools using antibody specific to the transcription factor PU.1 was also determined since V5 epitope tag. Specific antibody to PU.1 was used to determine this protein is critical for myeloid development. the level of protein expression in stable pools

Oncogene Runx1 regulation of macrophage growth and survival SR Himes et al 5284 Runx1/CBFb expression did not alter the level of PU.1 that are capable of self-renewal. As previously discussed, protein expression and was consistent with no observed Runx1 plays a clear role in suppression of growth during changes in EM-3 cell development (Figure 6d). lineage progression. Our data showing stable expression of Runx1 increased the number of EM-3 cells in G1/G0 is consistent with an ability to inhibit cell cycle Discussion progression. Although both loss of Runx1 and expres- sion of Runx1-ETO enhance self-renewal of progenitors, Control of CSF-1 receptor expression is critical for normal Runx1-ETO expression has a distinct effect on myeloid regulation of macrophage differentiation, proliferation cell differentiation, suppressing colony formation in and survival. When macrophages are deprived of CSF-1, response to cytokine treatment (Okuda et al., 1998; they respond by increasing transcription of c-fms mRNA Mulloy et al., 2002). Our results are consistent with resulting in enhanced expression of CSF-1 receptor on the these findings in that Runx1 activated an enhancer of c- cell surface. If there is a progressive loss of receptor fms gene expression but was not required for minimal signaling, cells will undergo programmed cell death. The expression through the promoter. The RAW264.7 cells data presented here extend previous studies by showing expressed CSF-1 receptor at low levels despite unde- that Runx1 is both a critical regulator of c-fms gene tectable levels of Runx1 expression. The ETO histone expression and a member of a novel class of genes that is deacetylase activity in the fusion protein must then act specifically induced following growth factor deprivation. to dominantly suppress promoter trans-activation in We have shown previously that interactions between mice expressing Runx1-ETO. the FIRE downstream enhancer within intron 2 and the A second family member, Runx3, has similar tumor promoter were required for c-fms gene expression in suppressor properties to Runx1. Loss of Runx3 function macrophages (Himes et al., 2001). Previous studies on is causally related to progression of gastric cancer in the function of Runx1/AML in humans have focused mice and humans (Li et al., 2002). Consistent with our solely on the promoter (Zhang et al., 1996a), although Runx1 expression studies, ectopic expression of Runx3 recently Follows et al. (2003)confirmed that putative in gastric cancer cell lines resulted in inhibition of cell runx1/AML1 sites within FIRE are occupied in both growth (Fukamachi and Ito, 2004). Runx1 and Runx3 mouse and human genes. CBF proteins function through bind similar sequences and both form protein complexes their ability to bind multiple families of transcription with the cofactor CBFb; hence, both proteins may factors and transcriptional cofactors. The postulated role inhibit growth by activating expression of common of the CBF protein complex as an organizing factor was tumor suppressor genes (Levanon et al., 2003). Runx3 supported by the highest level of Runx1/CBFb induction mRNA was also detectable in mature macrophages occurring when the enhancer was located distal to the (data not shown)and this expression may be involved in promoter (Figure 3). This observation may, in part, the ability of CBFb expression alone to effect cell explain the lack of conservation in the position of CBF growth and survival. Identification of genes whose binding sites in the promoters and enhancers of the expression can be activated by either Runx1 or Runx3 mouse and human genes. could provide an insight into the reduced capacity for Runx1 expression in mature macrophages showed an self-renewal during myeloid lineage progression. inverse correlation to the level of cell proliferation and The data presented here and by others suggest survival. Removal of CSF-1 results in a rapid cell cycle that CBF transcription factors function as an important arrest and a substantial rise in the rate of cell death. The link between cell differentiation and proliferation or corresponding increase in the level of Runx1 protein survival. Runx1 may then regulate genes that both could lead to expression of genes that control both promote differentiation of progenitor cells and reduce cellular proliferation and survival. This possibility is their capacity for self-renewal. CSF-1 receptor levels supported by the ability of Runx1 to trans-activate increase as myeloid cells differentiate to macrophages expression of p14ARF, a potent tumor suppressor that and Runx1 may be an important inhibitor of cytokine- can activate p53 by inactivating Mdm2 (Linggi et al., independent growth or survival in mature macrophages. 2002). Runx1 expression in response to growth factor High-level expression in mature macrophages may then withdrawal may, in part, act by raising the level of provide a terminal differentiation signal that blocks cell p14ARF. Expression of the CBFb-SMMC translocation proliferation. product in p16(INK4A)/p19ARF(À/À)mice resulted in increased development of acute leukemia suggesting that Runx1 regulates additional tumor suppressor genes to Materials and methods p14ARF (Yang et al., 2002). The resistance of EM-3 cells and sensitivity of pro- Cell culture and transfection monocytic cell lines to ectopic expression of Runx1 was consistent with distinct regulation at late stages of Bone marrow cells were isolated from the femurs of adult C57/ black 6 mice and cultured in RPMI media containing macrophage development. CML is a malignant disorder penicillin–streptomycin antibiotics (Gibco/Invitrogen). Cells of hematopoietic stem cells characterized by a prefer- were differentiated to macrophages by a 5-day treatment with ential expansion of myeloid cells resulting in high blood 1000 units of human CSF-1 (Chiron Corp., Emeryville, CA, counts and splenomegaly (Sawyers, 1999). Runx1 is USA). RAW264.7, Mono-Mac-6 (ATCC) and EM-3 cells normally expressed in hematopoietic progenitor cells (DSMZ)were cultured in RPMI medium as described above.

Oncogene Runx1 regulation of macrophage growth and survival SR Himes et al 5285 The pGL0.5fms, pGL0.5fmsDE and pGL0.5fmsUE plasmids cultured for 48 h before treatment with 1 mg/ml MTT (3-(4, 5- have all been previously described (Himes et al., 2001). The dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) in pEFRunx1 and pEFCBFb plasmids were made by RT–PCR 50 ml of media for 1 h. Cells were then treated with isopropyl amplification from mRNA using specific primers for insertion alcohol and absorbance at 595 nM measured. For cell cycle into the pEF6 vector in frame with a C-terminal V5/His tag analysis, 2 Â 106 transfected EM-3 cells were cultured in 25 cm2 (Invitrogen). The integrity of the coding region was confirmed flasks for 24 h in fresh media before harvesting and fixation in by DNA sequencing and protein expression verified by 70% ethanol. Fixed cells were stained with propidium iodide Western blot of V5 tagged recombinant protein in cell lysates according to established protocols. Cells were analysed for after transient transfection. Stable transfections were per- propidium iodide fluorescence on a Facstar flow cytometer formed by electroporation of 5 Â 106 cells in 400 ml RPMI (Becton Dickinson). media containing 20 mM HEPES and 10 mg of expression plasmid at 280 V and 1000 mF capacitance on a Bio Rad Gene Pulser (Bio Rad), followed by selection with 1 mg/ml Blas- Western blot and immunofluorescent stain tocidin. Transient transfections were performed as above with Protein from nuclear extracts was desalted on microcon 5 mg of reporter plasmid and 10 mg of expression plasmid. All columns and 5 mg of protein denatured for resolution on pre- reporter transfections were performed using a common pool of cast 12% polyacrylamide gels (Invitrogen)before blotting onto cells since no significant changes in transfection efficiency were PVDF membrane. Blots were stained with specific antibody to observed when measured using a CMVGFP expression Runx1, PU.1 (Santa Cruz Biotechnology)or V5 epitope (Cell plasmid in parallel transfections and flow cytometry. As in Signalling)and bands visualized using a horseradish perox- previous studies (Himes et al., 2001). we have chosen not to use idase conjugated antibody to goat IgG (Pierce), rabbit IgG or an internal reference promoter because changes in reference mouse IgG (Cell Signalling)and chemiluminescence substrate promoter activity can alter the test promoter through (Amersham). Cell surface expression of CSF-1 receptor was promoter competition. Cells were lysed 36 h post-transfection measured by indirect immunofluorescence. Cells were stained and assayed for luciferase activity according to the manufac- with CSF-1R-specific monoclonal antibody (Sudo et al., 1995) turer’s protocol (Roche Biochemical). The concentration of or isotype control (Cochet et al., 1998)followed by a FITC protein was determined by BCA protein assay (Pierce)and the labelled conjugate antibody (Serotec)and fluorescence levels level of luciferase activity was given as relative light units measured by flow cytometry. (RLU), calculated as light units/mg of protein assayed.

Electrophoretic mobility shift assay RNA isolation and quantitative PCR Isolation of nuclei and extraction of protein from BMM was Total RNA was prepared using RNeasy isolation kits (Qiagen) performed according to established protocols and the con- according to the manufacturer’s instructions. RNA was centration of protein in nuclear extracts was determined by treated with DNase 1 (Ambion)and reverse transcribed to BCA protein assay (Pierce). The double-stranded oligonucleo- cDNA using Superscript reverse transcriptase (Life Technol- tide probes used contained the following sequences: HRxP, ogies). Negative control samples (no first strand synthesis) CCAAACTCTGTGGTTGCCTT; MRxP, CCAAATTCTG were prepared by performing reverse transcription reactions in TAGTTCCCTT; MRxE, GCTGACACCACACAGGCAAC the absence of reverse transcriptase. cDNA levels of murine GA; HMRxE, TTTCCGCCCACACAGGCCG; PEBP2, hypoxanthine phosphoribosyl transferase (HPRT), Runx1 and GGATATCTGTGGTAAGCA. Binding reactions contained CSF-1R were quantitated by SYBR green, real-time PCR 20 mM HEPES buffer pH 7.4, 40 mM NaCl, 15% glycerol, 1 mg using an ABI Prism 7700 sequence detector (Applied of protein from nuclear extract, 25 ng poly(dI-dC)and Biosystems)according to the manufacturer’s instructions. approximately 0.01 pmol of 32P-labelled probe for 20 min at Amplification was achieved using an initial cycle of 501C for room temperature. Antibody supershifts were performed by 2 min and 951C for 10 min, followed by 40 cycles of 951C for preincubating binding reactions with antibody to Runx1 15 s and 501C for 1 min. Specificity of PCR was checked by gel (Santa Cruz Biotechnology)for 15 min at 4 1C before addition electrophoresis and melting curve analysis. cDNA levels of probe and protein binding. Competitions were performed during the linear phase of amplification were normalized by addition of unlabelled oligonucleotide immediately before against HPRT controls. Determinations were made in 7 addition of labelled probe. Protein binding was resolved on triplicate and mean s.d. was determined. Real-time primers 5% mini-polyacrylamide gels (Bio Rad)in 0.5XTBE (45 m M (f, forward; r, reverse)used to detect expression of the 0 Tris base, 45 mM boric acid and 1 mM EDTA)for 1 h at 100 V. corresponding murine genes were as follows: Runx1: f, 5 -CA CCGACAGCCCCAACT-30;r,50-CCCCAGTGCCACCACCT- 30; CSF-1R: f, 50-TACCACCATCCACTTGTATGT-30,r,50-TG Analysis of cell cycle and apoptosis TCCTCCACTGTCACCAACTC-30;HPRT:f,50-GCAGTA For MTT assays, 1 Â 104 transfected cells/well (viable by CAGCCCCAAAATGG-30;r,50-AACAAAGTCTGGCCTG trypan blue exclusion)were seeded into 96-well plates and TATCCAA-30.

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