Complex regulation of acetylcholinesterase gene expression ... - Nature

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Oncogene (2002) 21, 8428 – 8441 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Complex regulation of acetylcholinesterase gene expression in human brain tumors Chava Perry1,2, Ella H Sklan1, Klara Birikh1,4, Michael Shapira1,5, Leonor Trejo3, Amiram Eldor2,6 and Hermona Soreq*,1 1

Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel 91904; Department of Hematology, The Tel-Aviv Sourasky Medical Center, Tel Aviv and Tel-Aviv University, Israel 64239; 3Department of Pathology, The Tel-Aviv Sourasky Medical Center, Tel Aviv and Tel-Aviv University, Israel 64239; 4Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, 117799, Russia

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To study the regulation of acetylcholinesterase (AChE) gene expression in human brain tumors, 3’ splice variants of AChE mRNA and potentially relevant transcription factor mRNAs were labeled in primary astrocytomas and melanomas. AChE-S and AChE-R mRNA, as well as Runx1/AML1 mRNA accumulated in astrocytomas in correlation with tumor aggressiveness, but neither HNF3b nor c-fos mRNA was observed in melanoma and astrocytomas. Immunohistochemistry demonstrated nuclear Runx1/AML1 and cellular AChE-S and AChER in melanomas, however, only AChE-S, and not the secreted AChE-R variant, was retained in astrocyte tumor cells. Runx1/AML1 revealed weak linkage with ACHE promoter sequences, yet enhanced ACHE gene expression in co-transfected COS1 cells. The p300 coactivator and the ACHE promoter’s distal enhancer facilitated this effect, which was independent of much of the Runx1/AML1 trans-activation domain. Surprisingly, GASP, a fusion product of green fluorescence protein (GFP) and ASP67, a peptide composed of the 67 Cterminal amino acid residues of AChE-S, localized to COS1 cell nuclei. However, GARP, the corresponding fusion product of GFP with a peptide having the 51 Cterminal residues of AChE-E or GFP alone, remained cytoplasmic. Runx1/AML1 exhibited improved nuclear retention in GASP-expressing COS1 cells, suggesting modulated nuclear localization processes. Together, these findings reveal brain tumor-specific regulation of both expression and cellular retention of variant ACHE gene products. Oncogene (2002) 21, 8428 – 8441. doi:10.1038/sj.onc. 1205945 Keywords: acetylcholinesterase; human brain tumors; Runx1; AML1

*Correspondence: H Soreq; E-mail: [email protected] 5 Current address: Department of Genetics, Stanford University School of Medicine, CA, 94305-5120, USA 6 Deceased Received 6 May 2002; revised 31 July 2002; accepted 7 August 2002

Introduction Acetylcholinesterase (AChE) is expressed in brain tumors including meningiomas, astrocytomas and glioblastomas (Barbosa et al., 2001; Razon et al., 1984; Saez-Valero and Vidal, 1996). While this is compatible with the ubiquitous nature of AChE expression patterns, the molecular origin(s) of AChE’s regulation in brain tumors are still unknown. AChE expression in normal brain is regulated by c-fos and HNF3b, under various stress conditions (Kaufer et al., 1998; Meshorer et al., 2002; Shapira et al., 2000). However, it remained unclear whether the same transcription factors control ACHE gene expression in malignant brain cells, which of the AChE alternative splicing products is expressed in these tumors and what is the subcellular distribution of their corresponding protein products. Answers to these questions are an essential pre-requisite for understanding the yet obscure morphogenic function(s) of AChE in brain tumors. Human AChE enhances proliferation and/or differentiation of cultured glioma, neuroblastoma, osteosarcoma and phaeochromocytoma cells as well as of primary hematopoietic progenitor cells and dorsal root ganglia (Bigbee et al., 1999; Koenigsberger et al., 1997; Soreq and Seidman, 2001). To initiate an indepth study into this subject, we searched for the basic principles governing ACHE gene expression in human brain tumors. As a model, we selected astrocytomas and the more aggressive glioblastoma tumors, which usually develop in pre-existing astrocytomas (Barbosa et al., 2001; Razon et al., 1984). These tumors account for 50% of all brain tumors and 80 – 90% of adult glial tumors. Astrocytomas are graded into several subclasses of increasing invasiveness associated with distinct morphologies (Daumas-Duport et al., 1988), which may assist in defining their cellular properties. Nevertheless, the pathogenesis of glial brain tumors is not well understood, and genes and proteins that are involved in brain tumor proliferation are continuously sought. Interestingly, AChE levels are very low in all types of normal glia, but increase in astrocytic tumors (Karpel et al., 1996; Soreq et al., 1984). This hinted at the possibility that tumor-specific transcription factors

Regulation of ACHE expression in human brain tumors C Perry et al

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regulate ACHE gene expression in astrocytomas. For comparison, we used metastatic brain melanoma. The ACHE promoter The ACHE promoter includes a proximal 600 bp region essential for ACHE gene expression in benign tissues (Ben Aziz Aloya et al., 1993; Getman et al., 1995), but which, in transgenic mice failed to induce detectable expression in non-neuronal cells (Beeri et al., 1995). The proximal promoter includes, among others, consensus motifs for the leukemia-associated factor AML1/Runx1 (Perry et al., 2002), and c-fos, a transcription factor known to regulate ACHE gene expression under stress (Kaufer et al., 1998). A distal enhancer domain of the human ACHE promoter, located 17 Kb upstream from the ACHE transcription start site, includes functional binding sites for osteogenic factors such as 17b-estradiol and 1,25-dihydroxy vitamin D3, two adjacent functional binding sites for HNF3b, a glucocorticoid-response-element and binding sites for NF-kB, c-fos and C/EBP (Shapira et al., 2000). Transcriptional control of AChE production depends also on an internal enhancer positioned within the first intron, which contains consensus-binding sites for AP2, SP1, NF-kB and GATA1 (Chan et al., 1999). All of these areas in the ACHE locus were thus considered as potentially capable of directing ACHE gene expression in brain tumors. AChE has three 3’ splice variants that exert distinct activities The principle AChE mRNA transcript in brain and muscle tissues, which encodes AChE-S, the ‘synaptic’ isoform, is formed by joining exon (E) 4 to E6 to give rise to an amphipathic C-terminus. The second most common transcript which translates into AChE-E, the ‘erythrocytic’ isoform, links E4 and 5 and encodes a 43 amino acid C-terminal peptide. Cleavage after amino acid 14 of the open reading frame in E5 enables linkage to glycosylphosphatidyl inositol, integration and thus anchorage to erythrocyte membrane surfaces (Haas et al., 1996), however, this transcript is insignificantly expressed in the brain (Soreq et al., 1984). A third transcript, normally expressed in embryonic and tumor cells (Karpel et al., 1994), is also overproduced in response to psychological stress, under AChE inhibition or following head trauma (Kaufer et al., 1998; Shohami et al., 2000). Intron 4 encodes, in this case, the hydrophilic C-terminal extension of 26 amino acid residues of the ‘readthrough’ AChE-R (Li et al., 1991). When over-expressed in rat glioma, the AChE-S transcript, induced a differentiated astrocyte phenotype, while the AChE-R transcript yielded small, round cells (Karpel et al., 1996). ARP, a synthetic peptide derived from the AChE-R C-terminus, but not ASP, the AChE-S C-terminal peptide, induces cell proliferation of human hematopoietic progenitors (Grisaru et al., 2001). Therefore, it was of interest to compare the expression levels, the cell proliferation effects and the

subcellular localization of these two variant products of the ACHE gene in brain tumors. Here, we report the expression of the S and R variants of AChE, as well as of Runx1/AML1, a major hematopoietic transcription factor, in astrocytomas of increasing aggressiveness and in brain metastases of malignant melanoma. Our findings implicate Runx1/AML1, known to be involved in human leukemia (Perry et al., 2002; Westendorf and Hiebert, 1999), in the control of ACHE gene expression in human brain tumors and suggest a re-enforcement paradigm whereby these two intriguing proteins affect each other’s properties. Results Several transcription factor binding sites along the ACHE promoter were considered as potential regulators of its expression in human brain tumors. These included consensus binding sites for HNF3a/b and cfos in the distal upstream enhancer domain (Shapira et al., 2000) and for c-fos and Runx1/AML1 in the proximal promoter and first intron (Figure 1A). c-fos and HNF3b were both shown to control neuronal AChE expression under psychological and chemical stresses, respectively (Kaufer et al., 1998; Shapira et al., 2000). However, to the best of our knowledge, their expression was never sought in astrocytes. Runx1/ AML1, a major hematopoietic transcription factor is known to be associated with human leukemia (Perry et al., 2002; Westendorf and Hiebert, 1999), but was not considered to exist in brain tumors. First, we tested the functional relevance of these factors for controlling AChE gene expression in astrocytomas by in situ hybridization. Runx1/AML1 and AChE are co-over-expressed in primary brain tumors High resolution in situ hybridization revealed very low expression levels for the AChE-S and AChE-R transcripts in paraffin sections from grade I primary astrocytomas, consistent with the low level of ACHE gene expression in non-tumor astrocytes (Soreq and Seidman, 2001). In contrast, the labeling intensities of both these transcripts increased significantly in tumors with increasing aggressiveness (ANOVA: P50.0001) (Figure 1B). The labeling difference in five sections from 4 – 5 different tumors in each grade was especially large for AChE-S between astrocytoma grade I and II (P5161079, Student’s t-test), with a mild increase from grades II to III (P50.08). For the AChE-R transcript, differences grew to be more significant with increasing grade (P50.0005 for both I to II and II to III, but 761077 for III to IV), suggesting that the mechanisms controlling alternative splicing of AChEmRNA are related to tumor aggressiveness. cfos and HNF3b mRNAs, both known to be coexpressed with AChE in neurons, were not significantly expressed in astrocytomas, although c-fos was weakly detected in grade IV astrocyte tumors (Figure 1B). Oncogene


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Figure 1 Co-overexpression of Runx1/AML1 and AChEmRNA transcripts in astrocyte tumors. (A) Transcription factor binding sites and alternative splicing options in the ACHE locus: depicted is the reverse sequence of the cosmid insert (accession no. AF002993) containing the ACHE gene and 22 kb of its upstream sequence. Exons (numbered above) and introns (numbered below) are marked. Arrows designate positions of the distal enhancer domain (D.D.), the proximal promoter (P.P.) and the intronic enhancer (I.E.) along the cosmid reverse sequence (nt 22,465 being the ACHE transcription start site). Consensus binding sites for the noted transcription factors are represented by wedges and stars above. (B) Expression of AChE variants and various transcription factors in astrocytoma and metastatic brain melanoma. Presented are results of in situ hybridization using 5’-biotinylated cRNA probes selective for the AChE-S and AChE-R mRNA variants as well as for the Runx1/AML1, c-fos and HNF3b mRNA on sections of human astrocytomas at various pathological grades and metastatic brain melanoma

Surprisingly, Runx1/AML1 mRNA levels, detected by in situ hybridization, increased with astrocytoma aggressiveness in a similar manner to that of the AChE mRNA transcripts (ANOVA: P50.0001). Two independent Runx1/AML1 probes revealed similar patterns in astrocytomas grade I to III, corroborating the significance of these findings (Figure 1B and data not shown). A steep increase (P50.0002, Student’s ttest) between grades I and II, an insignificant increase (P50.14) between II to III, and a decrease (P50.008) between III to IV, suggested potentially causal correlation between Runx1/AML1 and the graderelated pattern of AChE gene expression. In sections from brain metastases of malignant melanoma, AChES, AChE-R and Runx1/AML1mRNA were all expressed as intensively as in grade III – IV astrocytomas (Figure 1B). Runx1/AML1 and AChE overexpression in tumor-associated vascular endothelial cells The gradual elevation in astrocytoma ACHE gene expression could be limited to the tumor cells. Alternatively, it could be caused by soluble growth factors, in which case, it should also take place in surrounding non-tumor cells. To distinguish between these possibilities, we examined vascular endothelial cells from astrocytomas of increasing aggressiveness. Diffuse cytoplasmatic signals of AChE and Runx1/ Oncogene

AML1 transcripts increased within all of the examined vascular structures from astrocytoma and metastatic brain melanoma (Figure 2A). In contrast, HNF3b and c-fos showed negligible expression levels in vascular endothelium (data not shown), suggesting that similar elements induced Runx1/AML1 and ACHE gene expression in tumor astrocytes and adjacent endothelial cells. Image analysis quantified the AChE mRNA variants in astrocytes and endothelial cells. Figure 2B presents, for four tumor specimens from each pathological grade, tumor grade-related increases in AChE-S, AChE-R and Runx1/AML1, but not c-fos or HNF3b mRNA. AChE expression in endothelial cells also depended on tumor grade (ANOVA: P50.0001 for AChE-S, AChE-R and Runx1/AML1) (Figure 2C), however with more limited increases in expression levels than those observed in tumor cells. Endothelial AChE-S mRNA increased significantly from grade I to II (P50.0002, Student’s t-test) but not from II to III or III to IV; AChE-R mRNA increased less dramatically, even from grade I to II (P50.02), whereas Runx1/AML1 increased steeply from grade II to III (P50.002) but presented lower differences between grades III to IV (P50.03). Inter-specimen variations were relatively small, both in tumor cells and in vascular endothelium. Altogether, the levels of both AChE-S and Runx1/AML1 transcripts increased steeply with the early increase in astrocytoma aggres-


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Figure 2 Correlated expression patterns of AChE variants and Runx1/AML1 in astrocytoma tumors. (A) Expression pattern in endothelial vascular cells supplying astrocytoma and metastatic brain melanoma. Shown are representative tumor tissue sections including labeled vascular endothelial cells (arrows), following in situ hybridization. Experimental details were as in Figure 1B. (B and C) Shown are average mRNA labeling intensities (mean values+s.e.m.) as the percentage of red pixels, falling within a defined intensity range. (B) Malignant tumor cells. (C) Vascular endothelial cells supplying the tumor. (D) left: Mean values (+s.e.m.) of AChE-R mRNA plotted as a function of AChE-S mRNA labeling in tumor cells. (D) right: Labeling in vascular endothelial cells of astrocytoma grade I – IV. R2 values were calculated for best fit curve in each case. *Statistically significant increase, from the bar on its left Oncogene


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siveness, whereas AChE-R mRNA levels increased more steeply in tumors of higher grades. Increasing AChE mRNA splicing shift with tumor aggressiveness The association between labeling intensity of AChE-R mRNA and AChE-S mRNA could be best-fitted by an exponential curve that expressed increased ratios of R:S transcripts with tumor aggressiveness (r2=0.9852). This reflected an alternative splicing shift in tumor cells from AChE-S to AChE-R mRNA with increasing tumor aggressiveness. In contrast, endothelial cells presented a linear best-fit correlation (r2=0.9871, Figure 2D), demonstrating no alteration in the R : S ratio in near-by endothelial cells, in all tumor grades. Thus, AChE-R/AChE-S relationships were clearly different in tumor from the surrounding

benign cells. Changes in the levels of the different AChE mRNA transcripts do not necessarily reflect changes in transcription, as was reported by others (Coleman and Taylor, 1996). Therefore, the contrasting exponential correlation of the ratio between AChE-R and AChE-S expression in astrocytoma as compared with the linear correlation observed in endothelial cells called for further exploring the significance of this difference. Retention of Runx1/AML1 and AChE-S but not AChE-R in tumor astrocytes and adjacent cells In tumor cells, Runx1/AML1 labeling was pronounced and strictly limited to nuclei, attesting to staining specificity (Figure 3A1,B1). Immunocytochemistry detected increasing labeling intensities with antibodies targeted at ASP, the C-terminal peptide unique to

Figure 3 Runx1/AML1 and AChE variants detected in astrocytoma and metastatic brain melanoma. Shown are immunomicrographs of brain metastatic melanoma (A) and graded human astrocytoma (B) labeled with antibodies targeted at Runx1/AML1 (1, staining with DAB), the AChE-S C-terminal peptide, ASP and the AChE-R C-terminal peptide, ARP (2 and 3, respectively. Staining with Fast red). Arrowheads in B2 point at AChE-S nuclear localization in astrocytoma cells (C). Runx1/AML1 subcellular localization is cell type specific: (Left) Runx1/AML1 was immunodetected in tumor cell nuclei (white arrow), and in the neuronal cytoplasm (black arrow) in astrocytoma-adjacent regions. Neurons were identified due to their unique morphology that contains neuritic extensions. (Center) In metastatic melanoma Runx1/AML1 labeled endothelial cells in both nuclei and cytoplasm (arrow). (Right) In astrocytoma grade IV, Runx1/AML1 labeled both nuclei and cytoplasm of endothelial cells (arrow) Oncogene


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AChE-S, in astrocytoma tumors, especially grade III (Figure 3B2), and also in metastatic brain melanoma (Figure 3A2). In contrast, antibodies targeted at ARP, the C-terminal peptide of the soluble AChE-R protein, yielded barely detectable signals in astrocytoma sections (Figure 3B3), while producing intensive staining in metastatic brain melanoma (Figure 3A3). Tumor-specific mechanisms may thus confer translational inhibition or enable AChE-R secretion from and/or rapid degradation in astrocytoma cells but retain this protein in melanoma cells, while retaining cellular AChE-S in both tumors. Runx1/AML1 labeling was further observed in tumor-adjacent endothelial cells and neurons (Figure 3C), however, with different subcellular distributions. In over 95% of the tumor-adjacent endothelial cells, Runx1/AML1 presented cytoplasmic, in addition to nuclear, labeling. This could reflect impaired nuclear targeting and/or high cytoplasmic levels of the Runx1/AML1 protein, leading to its cytoplasmic retention in non-tumor cells. In neurons, Runx1/AML1 remained primarily cytoplasmatic (Figure 3C and data not shown), suggesting that its exclusive localization in the nucleus of tumor cells involves specific targeting. Over-expressed AChE-R induces glioblastoma cell proliferation The observed correlation between AChE’s level of expression and astrocytic tumor aggressiveness could reflect physiological processes that are significant to tumor growth or progression. To test this prediction, we transfected human glioblastoma U87MG cells with AChE-S or AChE-R expression vectors under CMV control and assessed the effect of these transfections on cell proliferation by measuring BrdU incorporation. AChE-R over-expression induced a 40+5% increase in cell proliferation in the transfected cells, significantly higher than the effect conferred by transfecting sham DNA (P50.001, Student’s t-test). In contrast, AChE-S induced a limited, insignificant increase of up to 10+5% in cell proliferation (P=0.5). Together with the increasing expression of AChE-R mRNA, as tumor aggressiveness increases, these findings are compatible with a variant-specific role for AChE-R in inducing astrocyte tumor growth. Runx1/AML1, and its co-activator p300, together facilitate AChE production To test the potential capacity of Runx1/AML1 to promote ACHE gene expression, we first performed electrophoretic mobility shift assays. These showed a weak association between Runx1/AML1 and consensus Runx1/AML1 binding sequences from the ACHE proximal promoter. The occurrence of a supershift in the presence of anti-Runx1/AML1 antibodies attested to the authenticity of this interaction (Figure 4A). Transfection studies into COS1 cells involved AChE constructs including the human proximal promoter and the first intron. To this we added the distal enhancer

domain, and the HNF3b binding site deletion, a naturally occurring polymorphism in ACHE’s distal enhancer domain (Shapira et al., 2000). Co-transfections included AChE constructs together with the full or truncated (t)Runx1/AML1 constructs, the latter devoid of a large part of its transcription activating domain, and a p300 vector (Eckner et al., 1994). Runx1/AML1 and p300 expression was under CMV promoter control. Catalytic activity measurements of AChE were used as a reporter, and COS1 cells transfected with a GFP expression vector served as control. Consistent with the weak interaction of Runx1/ AML1 with the ACHE promoter, there was an only minor increase (30+8% above baseline) in AChE activity in COS1 cells co-transfected with the proximal promoter construct AC6 and Runx1/AML1, as compared with cells transfected with AC6 alone (Figure 4B). Likewise, the Runx1/AML1 transcriptional co-activator p300 had no significant effect on AChE expression on its own. However, COS1 cells cotransfected simultaneously with the three vectors encoding AChE, Runx1/AML1 and p300 exhibited up to 300% enhanced AChE expression. This effect was increased up to sixfold, far higher than the transcriptional enhancement effect inflicted by HNF3b (Shapira et al., 2000), in the presence of the ACHE distal enhancer domain, either native or mutated (P40.03 for either p300 – wtAC6 – Runx1/AML1 or p300 – DAC6 – Runx1/AML1). The p300-induced effect was not altered by the absence of much of the Runx1/ AML1 trans-activation domain (tRunx1/AML1, Figure 4), similar to reports of others in HEK293 cells (Kitabayashi et al., 1998). Because the CMV promoter may also be controlled by p300, we tested whether the p300-induced difference in ACHE gene expression did not merely reflect an increase in Runx1/AML1 levels under these co-transfection conditions. However, immunoblot experiments revealed no apparent change in Runx1/AML1 levels under co-transfection with p300 (data not shown). This in turn, strengthened the notion of a p300 effect that is primarily assisted by the DNA binding domain of Runx1/AML1 in the proximal promoter. Further studies will be required to determine whether p300 intensifies the binding capacity of Runx1/ AML1 to the ACHE promoter and in what way the ACHE distal enhancer domain contributes to this interaction. ASP67, a peptide with the sequence of the C-terminal residues of AChE-S, facilitates Runx1/AML1 nuclear localization Alternative AChE mRNAs direct the synthesis of AChE isoforms with distinct C-termini, subunit structure and sub-cellular localization (Soreq and Seidman, 2001). Translation of AChE-S mRNA results in a protein with a C terminus containing a conserved CSDL motif involved in dimerization and cellular retention (Velan et al., 1994). Adjacent to this motif, AChE-S includes a unique, potentially helical sequence Oncogene


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P

E2-E6

P E1 E2-E6

P E1 E2-E6

Figure 4 Runx1/AML1-p300 co-transfection facilitates AChE expression in COS1 cells. (A) Runx1/AML1 protein interacts with the ACHE proximal promoter in gel shift assays. Presented are the results of electromobility shift gel assays using Runx1/AML1, extracted from reticulocytes and probes homologous to the Runx1/AML1 consensus binding sequence from the T cell receptor promoter (serving as control) and to Runx1/AML1 binding site from the ACHE proximal promoter. Mobility shift assays show a faint shifted probe band and a supershifted probe band where anti-Runx1/AML1 antibodies were added (upper arrows). (B) Co-transfection assays. Shown are fold increases of AChE activity in COS1 cells transfected with the AC6 vector (open bars), with added distal enhancer (wt, gray bars) or mutated enhancer (DAC6, black bars), either alone, or with p300, Runx1/AML1 or truncated t-Runx1/ AML1, alone or together with p300. All vectors included the minimal CMV enhancer-promoter, except for AChE vectors which contained the noted domains from the authentic human ACHE promoter. *Statistically significant increase, from the bar on its left. (Inset) AChE, Runx1/AML1 and p300 expression constructs used for transfection experiments. AChE distal enhancer domain, normal and mutated fragments are shown as dark gray boxes; mutated sequence is marked with a star. The proximal promoter (P) harbors a Runx1/AML1 DMEM consensus binding site (consensus motif below), followed by intron 1 (I1) and numbered exons (E). Runx1/ AML1 includes the DNA binding domain (black, BD-250 residues) and the trans-activation domain (white, TA), t-Runx1/AML1 encodes a truncated Runx1/AML1, lacking much of the trans-activation domain. p300 was as in (Eckner et al., 1994)

with apparent amphipathic properties (Figure 5B). In contrast, AChE-R mRNA translates into a hydrophilic protein with a C-terminal extension devoid of cysteine, which is therefore expected to remain monomeric and soluble (Figure 5D) (Grisaru et al., 1999b). These distinctions and the expression differences between the two AChE variants in astrocyte tumors called for testing the biological properties of the peptides derived from their characteristic C-terminal sequences. To this end, we constructed GFP-fused expression vectors with downstream AChE ‘synaptic’ peptide (GASP), complete with the putative helix and the conserved CSDL motif, a mutated GASP (mGASP) in which a frame-shift mutation changed the final 8 amino acid residues of this peptide, abolishing the CSDL motif, but retaining the helix, or the AChE-R ‘readthrough’ peptide (GARP) (Figure 5). Both monkey COS1 and rat PC12 cells transfected with a non-fused GFP expression vector showed widespread whole cell labeling (Figure 5A). In contrast, both normal and mutated GASP were found primarily in the nucleus (Figure 5B,C), suggesting that the putative amphipathic helix residues that are unique to ASP, induce its nuclear localization. Unlike GASP, GARP was totally confined to the cytoplasm (Figure 5D). The distinct subcellular localization of the two peptides, Oncogene

shown in cells originating from different species and tissues, suggests that the C-terminus-dictated targeting of the AChE variants may be broadly relevant. To test whether the nuclear localization of GASP could potentially affect nuclear transport mechanisms for other proteins, we performed co-transfection experiments with Runx1/AML1. When transfected alone, the Runx1/AML1 protein resumed nuclear localization in 70% of transfected COS1 cells, while in the remaining 30% it was found only in the cytoplasm. However, when COS1 cells were co-transfected with the Runx1/ AML1 and the GASP expression vector, Runx1/AML1 resumed exclusive nuclear co-localization. Its overlap with GASP (Figure 6) suggested that ASP, and perhaps AChE-S as well, may facilitate the nuclear localization of Runx1/AML1. Further studies will be required to determine the extent of this phenomenon in primary tumor cells and find out whether it involves direct ASPRunx1/AML1 interactions and if it affects other transcription factors. Discussion Our search for the regulation of ACHE gene expression in melanoma and astrocyte tumors demon-


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Figure 5 GASP but not GARP resumes nuclear localization in co-transfected COS1 and PC12 cells. (A – D) Shown are the GFP fusion products of the employed expression vectors. Hydrophobic amino acids in the C-terminal peptides, unique to each of the variants, are marked red. Predicted helix with amphipathic properties is drawn for ASP. N25 notes the common residues preceding the variant-specific peptide sequences. Also shown are representative confocal micrographs of COS1 and PC12 cells transfected with the noted vectors. Green fluorescence depicts the GFP fusion protein

strated a correlation of the shifted expression from the AChE-S to the AChE-R variant with the aggressiveness of primary astrocytomas. Moreover, a parallel increase in the expression of Runx1/AML1, and the enhancement of ACHE gene expression by Runx1/ AML1 and its co-activator p300 in transfected cells, suggested a role for these proteins in controlling ACHE gene expression in brain tumors. While the two tumor-expressed AChE variants were retained in melanoma metastases, the amphipathic AChE-S but not the soluble AChE-R was retained in astrocytic tumor tissues. Nevertheless, AChE-R over-expression promoted proliferation of cultured astrocytic cells. Intriguingly, the C-terminal peptide of AChE-S directed GFP into cultured cell nuclei and improved the nuclear targeting of Runx1/AML1. Together, these observations potentially reflect a re-enforcement mechanism whereby AChE-S assists the nuclear targeting or the nuclear anchoring of Runx1/AML1, which in turn enhances ACHE gene expression in brain tumors. Increased tumor aggressiveness then favors a splicing shift towards the AChE-R variant, which may potentiate cell proliferation.

The Runx1/AML1 gene The Runx1/AML1 gene is the most common target for chromosomal translocations in human leukemia, consistent with its key regulatory role in hematopoiesis. Disruption of the Runx1/AML1 gene prevented definitive hematopoiesis in fetal liver (Okuda et al., 1996), demonstrating that Runx1/AML1 is essential for terminal differentiation of blood cell lineages. Runx1/ AML1 also affects cell proliferation, migration and angiogenic tube-formation in cultured endothelial cells. Mice lacking the Runx1/AML1 gene die in utero because of central nervous system hemorrhage (Namba et al., 2000). Runx1/AML1B, the largest transcriptionally active isoform, is a relatively weak activator on its own. Compatible with our current findings, it often cooperates with various DNA binding factors as well as with non-DNA binding co-activators, to enhance transcription rates (Westendorf et al., 1998). Runx1/ AML1 products also associate with transcriptional coactivators, including p300/CBP, ALY and YAP (Kitabayashi et al., 1998; Perry et al., 2002). These do not bind DNA directly but stimulate transcription Oncogene


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the VEGF receptor Flt1, promote angiogenesis and increase tumor aggressiveness (Valter et al., 1999). Runx1/AML1B also contains at least three domains that can contribute to the repression of transcription through yet unknown mechanism(s) (Meyers et al., 1995). Thus, Runx1/AML1 may exert complex effects on gene expression in brain tumors. Runx1/AML1 regulation of AChE gene expression

Figure 6 GASP augments Runx1/AML1 nuclear localization. (A) Shown are representative confocal micrographs of COS1 cells transfected with the noted vectors (A). Green depicts the GFP fusion protein and red – Runx1/AML1. Yellow signals in merged pictures depict co-localization of both proteins. (B) Columns show percentage+s.e.m. of nuclear-labeled Runx1/AML1 in COS1 cells transfected with the noted expression vectors. Average of five co-transfection experiments

by acetylating histones and/or recruiting the RNA polymerase II transcription – initiation complex. Runx1/AML1 gene products bind DNA as a heterodimeric complex with CBFb which does not bind DNA but enhances the affinity of Runx1/AML1 to DNA (Look, 1997; Perry et al., 2002; Westendorf and Hiebert, 1999). The percentage of astrocyte tumor cells expressing the Runx1/AML1 activation partner Ets-1 correlates with tumor grade, and recurrent astrocytomas express significantly more Ets-1 than primary tumors (Kitange et al., 1999). Ets-1 expression is induced by vascular endothelial growth factor (VEGF), which is secreted by glioma cells and is involved in neoangiogenesis. VEGF-induced Ets-1 in glioma microvasculature endothelial cells, may trans-activate Oncogene

ACHE gene expression in neuronal and glial cells is affected and mediated by a variety of growth factors and cytokines, among them epidermal growth factor (EGF), nerve growth factor (NGF) and IL-1 (Acheson et al., 1984; Li et al., 2000; Yamada et al., 1996). The transcription factors c-fos and HNF3b were found to regulate ACHE expression under stress conditions in normal brain (Kaufer et al., 1998; Shapira et al., 2000). Our current findings point to p300 as a major element that potentiates the capacity of Runx1/AML1 to induce ACHE gene expression. The co-increase in ACHE variants and Runx1/AML1 further suggested that Runx1/AML1 regulates ACHE expression in astrocyte tumors and metastatic brain melanomas. In contrast, neither c-fos nor HNF3b, both known to affect neuronal ACHE expression under various stresses, were expressed in astrocyte tumors. Runx1/ AML1 labeling in human glial cells and neurons is consistent with the expression of its homologs in Drosophila neuroblasts (Duffy et al., 1991), zebrafish olfactory placodes and cells attached to the otic vesicles (Kataoka et al., 2000), possibly reflecting a yetunrecognized role for this transcription factor in the mammalian nervous system. Co-activation of ACHE gene expression by p300 in COS1 cells’ transfection assays, could reflect an indirect competition effect or a direct p300 – Runx1/AML1 interaction. In either case, however, this combined effect supports the notion that Runx1/AML1 controls ACHE expression in brain tumors in vivo. The transcription factors CBP and p300 are widely expressed, and are believed to regulate gene expression in most cell types. Paradoxically, CBP and p300 participate in both cell proliferation and tumorsuppressor pathways in a highly context-dependent manner. For example, p300 augments p53 and BRCA1-mediated transcription, but it also interacts with viral oncoproteins (adenovirus E1A, SV40 Tantigen and human papillomavirus E6 protein), that suppress p300-mediated transcription and contribute to transformation (Goodman and Smolik, 2000). The fact that p300’s effect became yet more significant in the presence of ACHE’s distal enhancer domain, suggests a potential involvement of that region in ACHE transcriptional regulation by Runx1/AML1. p300 – Runx1/AML1 enhanced AChE transcription in transfected COS1 cells, independently of much of the Runx1/AML1 trans-activation domain. This may reflect one or more of several possibilities: direct p300 – Runx1/AML1 interaction; exposure of ACHE promoter regions for more effective interaction with


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Runx1/AML1, through the histone acetyl transferase activity of p300; p300-dependent enhancement of Runx1/AML1 affinity to DNA, or an indirect mechanism involving additional, yet unknown components. In L-G murine myeloid progenitor cells, the fulllength trans-activation domain of Runx1/AML1 is essential for its interaction with p300. However, Runx1/AML1 truncation down to its N-terminal 294 residues barely affected binding to p300 in the human kidney epithelial 293 cells. Only when truncated down to 177 residues, Runx1/AML1-p300 interaction was abolished. Our current findings are hence compatible with the hypothesis that in certain cell types (or species), Runx1/AML1-p300 interactions are governed by amino acid residues out of the consensus transactivation domain (Kitabayashi et al., 1998). Tumor versus vascular cells The correlated increases in AChE and Runx1/AML1 transcripts with tumor aggressiveness support the hypothesis that soluble cytokines and growth factors affect both tumor cells and nearby non-malignant endothelial cells. As tumor aggressiveness increased, a shift in alternative splicing towards the ‘readthrough’ transcript was noticed in tumor cells but not in near-by endothelial cells, suggesting modified control over the splicing factors leading to AChE-R production in malignant, as opposed to benign cells. Shifted splicing is often associated with tumorogenicity; for example, metastatic melanoma cell lines lose their ability to express stem cell factor (SCF)-2 but retain their ability to express its alternatively spliced SCF-1 variant (Welker et al., 2000). Also, the VEGF 121 variant, when over-expressed in breast carcinoma cells, increased angiogenicity and tumorogenicity more than other VEGF splice variants (Zhang et al., 2000). Thus, the shifted composition of AChE variants may reflect a tumor-specific change in the balance of mRNA splicing, favoring the AChE-R variant. Further support to the notion that AChE-R may be the culprit in the development of aggressive astrocytoma comes from our in vitro cell proliferation assays where AChER was found to significantly potentiate human glioblastoma cell proliferation, while AChE-S had an insignificant effect. Variant-specific protein distribution Transcripts for both AChE variants were present in tumor cells. Both AChE-S and AChE-R were detected in metastatic brain melanomas, but the AChE-S protein was the only one to be retained within astrocytic tumor cells. AChE-R may have been either rapidly degraded or excreted from astrocytic tumor cells but not from melanoma cells; that it promotes proliferation of astrocytic cells supports the latter option. The complete absence of AChE-R from astrocytomas, but not melanoma, suggests tumor-type specificity of this process. Soluble AChE was previously observed in serum of patients with primary

carcinomas and human glioma (Saez-Valero et al., 1996; Zakut et al., 1988), where it may possibly serve as a surrogate marker for tumor aggressiveness. The GFP-fused AChE-S C-terminal peptide localized to the nucleus, while the GFP-AChE-R peptide was confined to the cytoplasm. Neither peptide includes a well-recognized nuclear localization signal (NLS), suggesting that the nuclear targeting depended primarily on structural, as opposed to sequence elements. Also, ASP67 nuclear localization was not affected by mutating its CSDL motif, known to be involved in its cytoplasmic retention (Velan et al., 1994), suggesting that its nuclear transport, unlike its cytoplasmic retention, depends on the 32 N-terminal residues of this peptide which include its potentially helical amphipathic domain. Moreover, ASP67 enhanced Runx1/AML1 nuclear targeting in COS1 co-transfected cells, somewhat improving the nuclear targeting that was found in COS1 cells transfected solely with the Runx1/AML1 expression vector. While this modest effect may be particular to the transfection assay, it may possibly reflect intensification of nuclear transport or nuclear anchoring processes under AChES overproduction, so that Runx1/AML1 functioning would be enhanced under co-expression with AChE-S. In such a re-enforcement mechanism, Runx1/AML1 would augment ACHE gene expression; AChE-S would facilitate Runx1/AML1 nuclear targeting/ anchoring, potentially promoting its transcriptional activities, and augmenting the tumorogenic process by activating other genes and AChE-R would enhance tumorogenic cell proliferation. The ACHE gene and malignancies ACHE gene modifications are common in leukemic patients (Lapidot Lifson et al., 1989), but, there is little information on such modifications in the wider population, and their consequences, if any, are not yet known. The long arm of chromosome 7, where the ACHE gene resides (Ehrlich et al., 1992; Getman et al., 1992), is often modified in acute myeloid leukemia (AML), chemotherapy-related myeloproliferative disorders (Le Beau et al., 1996), and tumors of neuronal and glial origin (Berger et al., 1985). Chromosome 7 anomalies in adult myelodysplasia and AML associate with poor prognosis (Grimwade et al., 1998). Also, anti-cholinesterase exposure, which enhances AChE gene expression (Kaufer et al., 1998), increases the risk of leukemia (Brown et al., 1990). The actual role of ACHE in tumorogenesis remains incompletely understood. Nevertheless, the previously reported morphogenic and proliferative properties of AChE, together with our current findings, may be clues to its role in astrocytic tumor development and aggressiveness. Over-expression of a gene in a tumor, per se, does not necessarily predict active involvement in tumorogenesis. However, correlated expression with tumor aggressiveness of genes known to promote cell proliferation, death or invasiveness, is more informaOncogene


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tive. For example, epidermal growth factor receptor (EGF-R), predictive of treatment responsiveness of metastatic breast cancer, significantly increases from stage I to IV in breast cancer patients (NeskovicKonstantinovic et al., 1999). Also, IL-6 gene expression, involved in cell growth and resistance to chemoand radiotherapy, was significantly higher in the aggressive glioblastoma than in other types and grades of glioma (Rolhion et al., 2001). Likewise, the matrix metalloproteinases MMP-13 and MT1-MMP increase with esophageal cancer aggressiveness (Etoh et al., 2000). In the case of ACHE, the observed correlation with astrocytoma aggressiveness initiated astrocytoma transfection experiments through which we have found a causal correlation between ACHE gene expression and the aggressiveness of astrocytic tumors. The molecular mechanisms involved in this effect include the previously unknown functioning of Runx1/AML1 and its co-activator p300 in regulating ACHE gene expression in astrocytic tumors, combined with modified alternative splicing, preferring the AChE-R variant, which we found to induce glioblastoma cell proliferation. Materials and methods Sequence data analysis Transcription factor binding sites were sought in a cosmid clone (GenBank accession no. AF002993) that spans the human (h) ACHE gene and 22 Kb of its upstream sequence, using the MatInspector 2.0 program (Quandt et al., 1995) (core similarity of 1, matrix similarity of 0.85) and Find – Patterns, of the University of Wisconsin software package. Primary tumor sections Formalin-fixed and paraffin embedded surgical specimens, were obtained with the authorization of the Tel Aviv Sourasky Medical Center’s Committee for Human Experimentation (according to the regulations of the Helsinki Accords). Sections of astrocytoma tumors (5 mm) were stained with hematoxylin-eosin and clinically graded according to the St. Ann-Mayo grading system (grades I – IV), based on nuclear atypia, mitoses, vascular proliferation and necrosis (Daumas-Duport et al., 1988). In situ hybridization This involved 5 mm paraffin sections and 5’-biotinylated, 2’O-methylated cRNA probes selective for ‘synaptic’ (AChE-S mRNA) and the ‘readthrough’ transcript (AChE-R mRNA) (Grisaru et al., 1999a). Additional probes included: Runx1/ AML1 mRNA (accession number NM009821); 5’-CGAGUUGCUAUGGCUGCCCUCGGUCUCCACCACGUCGCUCUGGCUGGGGA-3’; 5’-GGGUGAAAUGGGUGUCGCUGGGUGGACAGAGGAAGAGGUGAUGGAUCCCA-3’ c-fos mRNA (accession number V00727); 5’-CUGACACGGUCUUCACCAUUCCCGCUCUGGCGUAAGCCCCAGCAGACUGG-3’ and rHNF3b mRNA (accession number NM012743.1); 5’-UUCAUCCCCUGGCUGGCGUUCAUGUUGCUCACGGAAGAGUAGCCCUCAGG-3’. Staining was with alkaline phosphatase/streptavidin conjugate and Fast Red (Roche Diagnostics GmbH, Mannheim, Germany) Oncogene

as a substrate. Counterstaining of nuclei was with hematoxylin (Sigma, St. Louis, MO, USA). Image analysis and signal quantifications Images were captured in a Zeiss Axioplan microscope, equipped with a digital camera. Red stained areas in cytoplasmic regions of scanned images were quantified, using Adobe Photoshop 4.0 (Adobe Systems, Inc. San Jose, CA, USA) at 240 output levels. Background values were subtracted and findings expressed as mean+standard error of the mean for 10 – 20 representative cells, per sample (4 – 6 samples from different patients per grade). Tumor cells were stained at intensities which were found to be rather uniform for each tumor grade, allowing us to evaluate a group of representative cells for each tumor type. Variance analysis was with ANOVA as well as with Student’s t-test. Immunocytochemistry Performed, as previously described (Sternfeld et al., 2000), on 5 mm formalin fixed and paraffin embedded sections of astrocytoma tumors and metastatic brain melanomas, using rabbit antisera against AChE-S, AChE-R and the Runx1/ AML1 protein, with secondary antibodies conjugated to horseradish peroxidase. Transfected COS-1 cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed in phosphate-buffered saline (PBS), incubated in 0.1 M glycine buffer (in PBS, 10 min room temperature), dried overnight, washed twice with PBS and further handled as for tissue immunohistochemistry. Antibodies and working dilutions Polyclonal goat anti-human ASP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) 1 : 100; polyclonal rabbit antihuman ARP (Sternfeld et al., 2000) 1 : 100, polyclonal rabbit anti-human Runx1/AML1 (Ben Aziz-Aloya et al., 1998) 1 : 250 (for both immunohistochemistry, and immunocytochemistry), biotinylated donkey anti-goat and donkey antirabbit (Chemicon, Temecula, CA, USA) 1 : 200, horseradish peroxidase conjugated goat anti-rabbit and donkey anti-goat (Jackson Immunoresearch Laboratories, West Grove, PA, USA) 1 : 200. Plasmid constructs AC6, WtAC6 and DAC6 – the ACHE expression constructs containing the proximal promoter, intron 1 and exons E2 – E6, without (AC6) or with a 200 bp fragment from the ACHE upstream distal enhancer (wild type (wtAC6), and mutated (DAC6) sequences) (Shapira et al., 2000). Runx1/AML1 and t-Runx1/AML1, encoding the intact and truncated (250 amino acid residues, lacking much of the trans-activating domain encoding sequence) murine Runx1/ AML1 protein (Ben Aziz-Aloya et al., 1998), were gratefully received from Dr Y Groner (Rehovot, Israel). p300, encoding the co-activator p300 (accession number U01877) was gratefully received from Dr RH Goodman (Portland, OR, USA). AChE-S, and AChE expression vector, encoding the synaptic form of AChE under CMV control, was previously described (Ben Aziz Aloya et al., 1993). AChE-R, and AChE expression vector, encoding the AChE ‘readthrough’ variant under CMV control, was previously described (Seidman et al., 1995).


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Mobility shift assays EMSA was performed using double-stranded (ds)DNA probes homologous to tested Runx1/AML1 binding sites from restricted parts of the ACHE proximal promoter. Prox168(+): 5’-GATCACGGTACTCTGCCACACTCCCACATG-3’ (30-mer); Prox168(7): 5’-AGGTCATGTGGGAGTGTGGCAGAGTACCGT-3’ (30-mer) dsDNA probes homologous to tested Runx1/AML1 binding sites from restricted parts of the T cell receptor a promoter served for control: 5’-GGATCCAACTGACCGCAGCTGGCCGTGCGAAGATCT-3’ (36-mer); 5’-AGATCTTCGCACGGCCAGCTGCGGTCAGTTGGATCC-3’ (36-mer) Consensus Runx1/AML1 binding sequences are marked in bold letters. Experiments were performed essentially as described elsewhere (Silverman et al., 1999). Briefly, 0.5 ng P-labeled dsDNA were incubated (2 h on ice) in a total volume of 30 ml of 0.25 mM DTT, 0.1 mM EDTA and 20 mg protein from Runx1/AML1-overexpressing retyculocytes extracts. Reaction products were electrophoresed in 5% polyacrylamide gels. Pre-incubation of protein extracts with the P-labeled dsDNA was followed by incubation with anti-Runx1/AML1 polyclonal antibodies (1 : 1000 dilutions, for 20 min in room temperature) for supershift assays. Expression constructs for fusion proteins Expression constructs for fusion proteins of GFP and AChE C-terminal peptides-pGARP, encoding a fusion protein of GFP and ARP51, was designed to include a fragment of the human AChE-R cDNA (nt 1796 – 1865 of hACHE, accession no. M55040, followed by nt 1 – 111 from the genomic hACHE I4 – E5 domain (accession no. S71129, stop codon in position 86)) cloned into the Bsp120I/XbaI sites of pEGFP – C2 (Clontech Laboratories, Palo Alto, CA, USA). pGASP, encoding a fusion protein of GFP and ASP67, the Cterminal peptide of the synaptic form of human AChE, includes a fragment of AChE cDNA (nt 1794 – 2001, accession no. M55040) inserted into the EcoRI – SalI sites of pEGFP – C2 (Clontech Laboratories, Palo Alto, CA, USA). This results in expression of the following peptides fused in frame to the C-terminus of GFP: pGARP PLEVRRGL RAQACAFWN RFLPKLLS ATGMQGPAGSGWEEGSGSPPGVTPLFSP. pGASP RPLEVRRGLRAQAC AFWNRFLPKLLSATDTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL. DGASP A fusion protein of GFP and a mutated C-terminal peptide of the synaptic form of human AChE, was created by inserting into the EcoRI – SalI sites of pEGFP – C2 (Clontech), a fragment of AChE cDNA (nt 1794 – 2001, accession no. M55040) with a frame-shift mutation changing the final 8 amino acid residues of the C-terminal sequence into: RLAAQTCD. Transfections CO1 and human glioblastoma U87MG cells were grown in humidified chambers in Dulbecco’s modified Eagle’s medium (Biological Industries, Beit Ha’emek, Israel) supplemented

with 10% fetal calf serum (FCS) and 2 mM L-glutamine at 378C, 5% CO2. PC12 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, 8% donor horse serum (DHS) and 2 mM L-glutamine. Transfections of the cells with 2 mg plasmid DNA per well were carried out using Lipofectamine Plus (Gibco BRL Life Technologies, Bethesda, MD, USA) according to the manufacturer’s instructions. AChE activity assays, which served as reporter for transcription efficacy, were performed in cell homogenates, prepared 24 h post-transfection in 0.01 M phosphate buffer (pH 7.4) containing 1% Triton X100, left on ice for 45 min, then centrifuged at 48C in 14 000 r.p.m. for 45 min, supernatant collected and kept in 7208C until used. COS1 cells were also co-transfected with either the GASP or GARP expression constructs together with the noted Runx1/AML1 and p300 constructs. PC12 cells were transfected with either the GASP or GARP expression constructs. Cell proliferation assay U87MG human glioblastoma cells, gratefully received from A Levitzki, Jerusalem, were grown in 48-well plates and were transfected with 0.4 mg DNA per well of either the AChE-R or AChE-S expression plasmids or the pEGFP-C2 (Clontech Laboratories, Palo Alto, CA, USA) plasmid, serving as sham DNA. Lipofectamine Plus (Gibco BRL Life Technologies, Bethesda, MD, USA) was used according to the manufacturer’s instructions. Cell proliferation was assessed by incorporation of 5’-bromo-2-deoxyuridine (BrdU) as previously described (Grisaru et al., 1999a). Confocal microscopy Confocal microscopy was used to evaluate expression and intracellular localization of studied proteins, using a Bio-Rad MRC-1024 confocal scanhead (Hemel Hempstead, Herts, UK) coupled with an Zeiss Axiovert 135 microscope equipped with a plan apochromat 406/1.3 oil immersion objective. GFP and Fast-Red were excited at 488 nm and emission was collected through a 525+20 nm filter for GFP and a 580 nm+16 nm filter for Fast Red. The confocal iris was set to 3 mm. Sections were scanned every 0.5 mm. Images were merged using the Image Pro Plus software (version 4.0, Media Cybernetics, Silver Spring, MD, USA).

Acknowledgements We are grateful to Drs Yoram Groner, Ditsa Levanon and Varda Negreanu (Rehovot) for the Runx1/AML1 vectors and antibodies, assistance with experiments and fruitful discussions; to Dr Richard H Goodman (Portland, OR, USA) for the p300 plasmid, to Dr A Levitzki (Jerusalem) for human glioblastoma cell lines, and to Dr David Glick (Jerusalem) for reviewing this manuscript. The study was supported by the U.S. Army Medical Research and Materiel Command (DAMD 17-99-1-9547) and by Ester Neuroscience Ltd. Chava Perry, M.D., is the incumbent of a basic research fellowship from the Israel Ministry of Health. The other authors wish to dedicate this manuscript to our co-author, Prof. Amiram Eldor, who died while we were preparing this paper for submission.

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