The gene encoding the granulocyte colony-stimulating factor ... - Nature

Oncogene (1998) 17, 503 ± 510  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc

The gene encoding the granulocyte colony-stimulating factor receptor is a target for deregulation in pre-B ALL by the t(1;19)-speci®c oncoprotein E2A-Pbx1 Wim BM de Lau1, Jolanda Hurenkamp1, Paul Berendes2, Ivo P Touw3, Hans C Clevers1 and Marc A van Dijk1 1

Academic Hospital Utrecht, Department of Immunology F03.821, PO-Box 85500, 3508 GA Utrecht; Erasmus University, Departments of 2Immunology, 3Hematology, Dr Molenwater plein 50, Rotterdam, The Netherlands

Approximately 25 ± 30% of childhood pre-B cell acute lymphoblastic leukemias (pre-B ALL) is characterized by the presence of a (1;19)(q23;p13.3) translocation. The presence of this translocation is generally accompanied by a poor prognosis. The chimeric gene resulting from this chromosomal rearrangement encodes a hybrid transcription factor, E2A-Pbx1. In an attempt to delineate the genetic cascade initiated by E2A-Pbx1, we sought to identify genes that are deregulated by this transcription factor in t(1;19) pre-B ALL. We show here that the gene encoding the granulocyte colonystimulating factor receptor (G-CSFr) is speci®cally upregulated in pre-B cells expressing E2A-Pbx1. GCSFr is also expressed in cell lines established from t(1;19) pre-B cell leukemia and on primary t(1;19) tumor cells, but not on control cells. These data indicate that G-CSFr gene is a target for deregulation by E2APbx1. Keywords: G-CSF receptor; cDNA subtraction; leukemia; chromosomal translocation

Introduction Many leukemias are characterized by speci®c chromosomal abnormalities, such as translocations. A signi®cant number of these result in the modi®cation of transcription factor function, either by fusing parts of these transcription factors to other proteins, or by altering their expression (Cleary, 1991; Drexler et al., 1995; Rabbitts, 1994). The hypothesis is that these altered transcription factors aect the expression of downstream target genes, whose products play a role in leukemogenesis. Although there has been a considerable amount of research aimed at elucidation of the biochemical properties of the modi®ed transcription factors, the identi®cation of downstream targets has proven dicult. We have been studying the molecular eects of the (1;19) translocation. In 1984, the t(1;19(q23;p13.3) was identi®ed as a non-random chromosomal

Correspondence: MA van Dijk Received 15 August 1997; revised 11 March 1998; accepted 11 March 1998

abnormality in 25 ± 30% of childhood pre-B acute lymphoblastic leukemia (pre-B ALL) (Williams et al., 1984). The t(1;19) joins the genes coding for two transcription factors, the E2A gene on chromosome 19 and the Pbx1 gene on chromosome 1. This results in the expression of a chimaeric transcription factor, E2A-Pbx1 (Kamps et al., 1990; Nourse et al., 1990). E2A-Pbx1 consists of the amino terminal 477 amino acids of E2A, which contains the transactivation domains (Massari et al., 1996; Quong et al., 1993) fused to the carboxyl terminus of Pbx1, containing a homeobox DNA-binding domain (Kamps et al., 1990; Nourse et al., 1990). We and others have shown that E2A-Pbx1 is a strong transcriptional activator whereas the Pbx1 protein, from which E2APbx1 derives its DNA-binding domain, does not activate transcription by itself (LeBrun and Cleary, 1994; Lu et al., 1994; Van-Dijk et al., 1993). Expression of E2A-Pbx1 in mice causes leukemia, which indicates that the fusion protein is indeed oncogenic (Dedera et al., 1993; Kamps and Wright, 1994). Hence, being a strong transcriptional activator and an oncoprotein, E2A-Pbx1 likely upregulates the transcription of downstream target genes outside of their normal context. Identi®cation of these genes may provide insight into the development of pre-B ALL and may identify novel targets for therapeutic intervention. To identify E2A-Pbx1 target genes, we have used Representational Dierence Analysis (RDA). This technique has originally been developed to isolate dierences between genomic DNA from tumor and normal tissue, allowing the identi®cation of tumor suppressor genes deleted from the tumor DNA (Lisitsyn et al., 1993). It has since been adapted for use with cDNA, permitting the identi®cation of genes that are exclusively expressed in one cell type (Hubank and Schatz, 1994). By performing RDA on human pre-B cells (Reh) stably or transiently expressing E2A-Pbx1, we have found that the expression of the gene coding for the granulocyte colony-stimulating factor receptor (G-CSFr) is speci®cally upregulated in E2A-Pbx1 expressing cells. G-CSFr is also expressed on cell lines derived from t(1;19) pre-B ALL, as well as on clinical t(1;19) ALL bone marrow samples. The ®nding that the oncogenic transcription factor E2A-Pbx1 upregulates G-CSFr expression suggests that aberrant expression of this receptor might have a role in the pathogenesis of pre-B ALL.

The G-CSF receptor is a target for E2A-Pbx1 WBM de Lau et al

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Results Identi®cation of G-CSFr as a target gene for E2A-Pbx1 We have constructed a human pre-B cell line (Reh-23s) which stably expresses E2A-Pbx1b (=P77E2APbx1, Kamps et al., 1991). The expression of E2A-Pbx1b was con®rmed by Western blotting, using a polyclonal antibody speci®c for the breakpoint region of E2APbx1 (Berendes et al., 1995), and an anti-E2A monoclonal antibody (Yae). As shown in Figure 1, Reh-23s cells express only the 77 kd E2A-Pbx1b protein whereas RCH-ACV and Sup-B27, two cell lines originating from t(1;19) positive pre-B ALL express both E2A-Pbx1a and E2A-Pbx1b. Reh cells do not express E2A-Pbx1, but do express E2A proteins, as indicated on the right-hand panel. The amount of E2APbx1b expressed in Reh-23s is slightly lower than the amount of E2A-Pbx1b expressed in Sup-B27 and RCHACV. These latter two cell lines express approximately ®vefold more E2A-Pbx1a than E2A,-Pbx1b. To identify E2A-Pbx1 target genes, we have compared the pattern of gene expression in these cells with that of untransfected Reh cells using RDA. To assess the extent of enrichment achieved during subsequent rounds of subtraction, we cloned difference products from round 2 and sequenced the inserts from 100 independent clones. 24 of the 100 clones contained DpnII fragments derived from the neomycin resistance gene which was used to establish the Reh-23s clone, but which is not expressed in untransfected Reh cells. This indicates that the products in this round have been dramatically enriched for genes expressed only in the E2A-Pbx1 expressing cell line. 1 of the 100 clones contained a fragment (nucleotides 2411 ± 2644, Fukunaga et al., 1990) derived from G-CSFr (Table 1). To prevent further enrichment for fragments derived from the neomycin gene, we included 1 mg of the neomycin fragments recovered in the 2nd RDA round into the driver pool and performed a 3rd round of RDA. We again cloned the products and sequenced 20 independent inserts. 13 out of these 20 clones contained DpnII fragments derived from G-CSFr mRNA (Table 1). The recovery of three independent fragments from

G-CSFr gene, constituting 465% of the DNA fragments recovered in the 3rd RDA round is a strong indication that this gene is indeed dierentially expressed. Although uniquely expressed in 23s cells, we did not recover fragments derived from E2A-Pbx1. The most likely reason for this is that during successive rounds of RDA the fragments derived from the E2A portion are removed because E2A is also expressed in wt Reh cells (see Figure 1). The fragments derived from the Pbx1 portion are likely removed by hybridization with fragments derived from the highly homologous Pbx2 and Pbx3 mRNAs. Pbx2 and 3 are expressed in Reh cells and in most other tissues (Monica et al., 1991 and data not shown). To con®rm the result obtained in the ®rst RDA, we repeated the cDNA subtraction procedure with transiently transfected cells. In these cells, which have been harvested 40 h after transfection and compared with mock-transfected cells, the chances of inadvertant genetic changes are very slight. Reh cells (107) were transfected with either pCDNA-E2A-Pbx1b (tester) or pCDNAI-amp (driver). Expression of E2A-Pbx1 was veri®ed by immunostaining with the E2A-Pbx1 breakpoint speci®c antibody (Berendes et al., 1995), which revealed approximately 10 ± 15% of the cells to be positive (not shown). After three rounds of RDA, the product was compared with the third round RDA product obtained in the ®rst subtraction procedure by agarose gel-electrophoresis (Figure 2a). The left lane contains the products obtained during the ®rst procedure, the right lane those obtained using transiently transfected cells. The two major bands migrate at the size of the fragments derived from GCSFr. Two of the three G-CSFr DpnII fragments recovered in the ®rst subtraction were also present in the third round material from the second subtraction (nt 287 ± 502 and nt 2411 ± 2644). Furthermore, we recovered an additional G-CSFr fragment (nt 697 ± 830) not recovered in the ®rst subtraction. To evaluate the extent of enrichment achieved in successive rounds of RDA, equal amounts of driver and tester material as well as material from rounds 1, 2, and 3 from both subtraction procedures were separated on an agarose gel and transferred to nitrocellulose. This blot was subsequently hybridized with the three radiolabeled GCSFr DpnII fragments recovered in the ®rst RDA procedure. As shown in Figure 2b, upper panel, a large increase of hybridization signal is observed in consecutive RDA rounds. Note that only two hybridizing bands are observed, because two of the three fragments dier by only 2 bp, and co-migrate on this gel. This indicates that these fragments become dramatically

Table 1 Recovered G-CSFr DpnII fragments RDA round 2 (100 clones sequenced) Recovered fragment Figure 1 Analysis of E2A-Pbx1 expression in cell lines. Each lane contains total cellular protein from 26106 cells from the cell lines indicated at the top of each panel. Primary antibodies: left panel: aE2A-Pbx1 breakpoint-speci®c mouse polyclonal antibody (Berendes et al., 1995) and right panel: Yae monoclonal antibody (Santa Cruz Biotechnology). E2A-Pbx1a and E2A-Pbx1b protein bands are indicated, as well as the position of the normal E2A proteins

2411 ± 2644 (233 bp)

RDA round 3 (20 clones sequenced) Recovered fragments

n

287 ± 502 (215 bp) 2411 ± 2644 (233 bp) 2644 ± 2857 (213 bp)

4 7 2

G-CSFr DpnII fragments recovered during RDA procedure #1. Indicated are nucleotide positions as used in Fukunaga et al. (1990). Between brackets are the sizes of the individual fragments (without adapters). To the right is indicated the number of times a given fragment has been recovered


enriched during subsequent rounds of RDA. The exposure of this blot is such that the signal derived from G-CSFr fragments present in the tester material is not visible. To verify the presence of G-CSFr-derived DpnII fragments in the tester material from RDA #1 and #2 we made a second Southern blot containing approximately ®ve times the amount of driver and tester material used in Figure 2b. This blot was again hybridized with a probe derived from the recovered G-CSFr fragments. Figure 2c shows that only the target material, derived from E2A-Pbx1 expressing cells, contains material hybridizing with the probe. During RDA, genes expressed in both driver and target cells should be removed. To verify this, we hybridized the blot from Figure 2b, upper panel with a b-actin cDNA probe. As shown in the lower panel of Figure 2b, the strong b-actin hybridization signal present in driver and tester lanes of subtraction #1 and #2 is lost during successive rounds of RDA, indicating that PCR fragments derived from common genes are eciently removed during successive rounds of RDA. These data show that in Reh cells, either stably or transiently expressing E2A-Pbx1, G-CSFr gene is switched on.

a

b

Expression of G-CSFr on t(1;19) cells

c

Figure 2 (a) Comparison of ampli®ed material obtained after 3 rounds of RDA starting from stably or transiently transfected Reh cells. Approximately 1.5 mg of DNA was separated on a 2.5% agarose gel containing 0.5 mg/ml ethidium bromide and bands were visualized by UV. (b) Southern blot containing starting material (tester and driver) as well as material from successive rounds of RDA (DP1, DP2, and DP3) obtained with cells stably or transiently transfected with E2A-Pbx1b. The

To con®rm the data obtained by RDA, we next investigated the induction of G-CSFr protein expression on Reh cells transiently transfected with pCDNAE2A-Pbx1b by ¯uorescence-activated cell sorting (FACS), using the LMM741 antibody directed against the extracellular domain of human G-CSFr. Figure 3 shows that expression of G-CSFr is not present on untransfected Reh cells, but becomes detectable on approximately 16% of the transfected Reh cells within 24 h, and the expression is stable until at least 48 h after transfection. Next we investigated whether G-CSFr is also expressed on cell lines derived from t(1;19) patient material. We tested two t(1;19) positive pre-B cell lines; Sup-B27 and RCH-ACV (Jack et al., 1986; Nourse et al., 1990, see also Figure 1). We also tested Reh cells and Reh-23s cells. Figure 4a shows that in both Sup-B27 and RCH-ACV a signi®cant number of cells express GCSFr. The minimum number of G-CSFr molecules that can be detected by FACS using this protocol is approximately 100 (IP Touw, unpublished data). This means that cells with higher than background fluorescence (indicatd by the horizontal line) contain more than 100 receptors per cell, high enough to elicit a functional response (see Avalos, 1996 for a review).

samples were separated further than the samples shown in (a). Top panel: as a probe we used three DpnII fragments from GCSFr isolated during this procedure. The sizes indicated to the right are inclusive the 24 bp adapters. Bottom panel: As a probe human b-actin cDNA was used. (c) Southern blot containing approximately ®ve times more driver (D) and target (T) PCR fragments than used for (b). Numbers at the bottom indicate RDA procedure #1 or #2. The blot was hybridized using a 32Plabeled probe derived from the G-CSFr fragments from round 3 from RDA #1 (Table 1). The blot was exposed for 48 h and hybridizing material was visualized using a Molecular Dynamics phosphorimager

505


506

Figure 3 Upregulation of G-CSFr expression on Reh cells. Reh cells were transiently transfected with E2A-Pbx1b and the expression of human G-CSFr was analysed by FACS before transfection and 24, and 48 h after transfection. % of G-CSFr-positive cells is indicated

a

b

Figure 4 (a) Analysis of human G-CSFr expression on t(1;19) cell lines. The cell line is indicated in the upper left-hand corner of each panel. Indicated is the population of cells showing a higher than background staining with G-CSFr antibody. The left and middle bottom panels show analysis of cells derived from t(1;19) pre-B ALL. As a control, the left two panels show FACS analysis of G-CSFr expression on myeloid 32D cells (upper panel) and 32D cells stably transfected with G-CSFr (lower panel). % of CCSFr-positive cells is indicated. (b) RT ± PCR analysis of G-CSFr mRNA expression in t(1;19) cell lines. Equal amounts of ®rst strand cDNA from 56106 cells from the indicated cell lines were ampli®ed using G-CSFr-speci®c (top panel) or b-actin-speci®c (lower panel) primers. Samples were ampli®ed 25, 30 and 35 cycles as indicated. The size of the PCR fragments is indicated


As a control for the speci®city of the LMM741 antibody, we included (myeloid) 32D cells, which do not express G-CSFr, and 32D cells stably transfected with human G-CSFr (Dong et al., 1993). These results indicate that LMM741 only recognizes a surface antigen (G-CSFr) present on the G-CSFr-transfected 32D cells, and does not crossreact with other antigens on wt 32D cells (Figure 4a, left upper and lower panel). To con®rm the FACS data of the t(1;19) cell lines, we analysed the expression of G-CSFr mRNA by RT ± PCR. As a positive control we included U937, a myeloid cell line expressing G-CSFr (Khwaja et al., 1993). The results are depicted in Figure 4b. To detect G-CSFr-expression, we used primers described by Fukunaga et al. (1990). The PCR fragment derived from U937 cells is very abundant, with a signal detected already at 15 PCR cycles (not shown) con®rming the presence of high amounts of G-CSFr transcripts in this cell line. G-CSFr-speci®c PCR product becomes detectable in Reh-23s cells at 25 cycles. In Sup-B27 and RCH-ACV cells it becomes detectable at 30 cycles. It is not detectable in Reh cells (see Figure 4), nor in mock transfected Reh cells (not shown). The identity of G-CSFr PCR product was conformed by DNA sequencing. To show that equal amounts of cDNA were present at the start of the PCR, a control PCR was performed using b-actinspeci®c primers (Figure 4b, lower panel). To determine whether these data can be extended to clinical ALL samples, we tested ALL cells from the bone marrow of two t(1;19) positive pre-B ALL patients for the presence of C-CSFr using FACS analysis. To ensure that cells staining positive with G-CSFr are indeed from the B-cell lineage, we also stained with mouse anti-CD19-FITC, which recognizes CD19, a molecule expressed only on B-cells. Figure 5 shows that both patient samples contain considerable numbers of G-CSFr-expressing CD19 positive cells. To examine if the expression of G-CSFr is indeed speci®cally upregulated in t(1;19) pre-B ALL, we examined ALL cells from the bone marrow of seven additional patients for G-CSFr expression. Six of these patients were diagnosed with non-t(1;19) B-cell precursor ALL, one was a B-ALL (sIgM+). All six B-cell precursor ALL were negative, but the B-ALL stained positive for G-CSFr (data not shown). Occasional G-CSFr-positive ALL have been reported previously (Matsushita et al., 1997), but have thus far not been associated with a speci®c translocation event. Our results indicate that G-CSFr is expressed in two t(1;19)-positive cell lines and in two t(1;19)-positive patient samples. Together with the data generated by the subtraction procedures, these data strongly support the hypothesis that the upregulation of G-CSFr expression in t(1;19) ALL is a consequence of the expression of the E2A-Pbx1 oncoprotein. Functional role of G-CSFr in t(1;19) pre-B ALL ALL cells often exhibit only a very limited proliferative capacity when cultured ex-vivo, even with supplementary cytokines (reviewed in Lowenberg and Touw 1993). To investigate the potential role for G-CSFr on t(1;19) leukemic cells, we have cultured puri®ed bone marrow blast cells from the three t(1;19) patients described above with several cytokines, in combination

507

Figure 5 Analysis of G-CSFr expression on fresh t(1;19) leukemic cells from two patients. Top three panels: patient 1, bottom three panels patient 2. Negative controls: (a) cells from patient 1 and 2 stained with total mouse IGG1-FITC to establish background staining for CD19. (b) Cells from patient 1 and 2 stained with streptavidine-PE to establish background staining for G-CSFr. The panels (c) show the results obtained with puri®ed bone marrow blasts obtained from two t(1;19) patients. Y-axis: G-CSFr expression, X-axis: CD19 expression. % of G-CSFr and CD19 double positive cells (top right quadrant) is indicated

with G-CSF. We have used IL-1 through IL-4, IL-6 and IL-7, as well as Flt3 ligand, stem cell factor, and GM-CSF. Cells from patient #2 exhibited a moderately (2 ± 3-fold) increased proliferation in response to a combination of G-CSF with IL-3, Flt3 ligand or Stem cell factor, but not when these factors were provided individually (data not shown). The other patient did not show a signi®cant increase in proliferation with any of the tested combinations. This could mean that t(1;19) cells require an additional growth stimulus not provided by the combinations tested, or that additional genetic events occurring during the conversion towards acute leukemia determine to an extent the cellular response to G-CSF. Discussion One of the key questions in leukemia, as well as in other forms of cancer, is the precise nature of the changes that take place in a cell upon the occurrence of the primary genetic lesion, such as the (1;19) translocation in pre-B


508

ALL. We have identi®ed the upregulation of G-CSFr expression as one of the events that occur in pre-B ALL cells upon the expression of the chimaeric t(1;19)speci®c oncoprotein E2A-Pbx1. G-CSFr is a singlechain cytokine receptor normally expressed on myeloid precursor cells and mature neutrophiles as well as in the placenta (Fukunaga et al., 1990). G-CSF is essential for myeloid dierentiation, since G-CSF knock-out mice exhibit severe neutropenia and disturbed myeloid dierentiation (Lieschke et al., 1994). G-CSFr transduces signals that regulate the proliferation, maturation and survival of myeloid progenitor cells. The membrane-proximal cytoplasmic region transduces proliferative and survival signals, whereas the membranedistal region transduces (myeloid) maturation signals (reviewed in Avalos 1996). In non-myeloid cells, the dierentiation signal is not functional but proliferative signaling is unimpaired, as has been demonstrated for BAF3 cells. BAF3 is a mouse pro-B cell line that is dependent on IL-3 for proliferation and survival. When these cells are stably transfected with G-CSFr, they can survive and proliferate on G-CSF instead of IL-3. Removal of the carboxy terminal domain of G-CSFr, responsible for myeloid dierentiation, has no in¯uence on the proliferative capacity of these cells, indicating that this signaling domain is only functional in the context of a myeloid cell (Dong et al., 1993). Furthermore, G-CSFr has been implicated in carcinogenesis. A subgroup of patients with severe congenital neutropenia have point mutations in the cytoplasmatic domain of the receptor which cause deletion of the Cterminal maturation domain. These patients are prone to develop myeloid leukemia, most likely due to disfunctional signaling events (Dong et al., 1995). A role for C-CSFr in solid tumors has also been suggested, as it is expressed on small cell lung cancer and bladder carcinoma cells. In these cells G-CSF signaling contributes to proliferation (Avalos et al., 1990; Tachibana et al., 1995). Together these data make it likely that disfunctional (i.e. out of context) G-CSF signaling plays a role in certain types of cancer. Our data indicate that G-CSFr expression is speci®cally upregulated in t(1;19) pre-B ALL. A further indication that G-CSFr is a target gene is the presence of a perfect E2A-Pbx1 concensus binding site (ATCAATCAA) in G-CSFr promoter, at position 71198 to 71189 relative to the transcription start (Seto et al., 1992). We and others have found that E2APbx1 speci®cally recognizes and activates transcription from this DNA sequence (LeBrun and Cleary, 1994; Lu et al., 1994; Van-Dijk et al., 1993). The element from GCSFr promoter is capable of mediating transcriptional activation by E2A-Pbx1 when present as a multimer upstream of a minimal promoter in a reporter assay (data not shown). However, in the context of the wildtype G-CSFr promoter this single site is not sucient in mediating transcriptional activation by E2A-Pbx1 (not shown). Although the presence of this site in G-CSFr promoter would seem to indicate that G-CSFr is a direct E2A-Pbx1 target gene, rather than the result of later events, we are at present unable to con®rm this. This could indicate that an additional, and currently unde®ned, G-CSFr enhancer region is required to mediate full transactivation. Alternatively, these results could indicate that G-CSFr is a target further downstream in the cascade initiated by E2A-Pbx1.

In normal bone marrow, colony stimulating factors such as IL-3, GM-CSF and G-CSF are required for suppression of apoptosis and enhancement of proliferation, and as such important, for regulation of the size of the normal hematopoietic precursor population in the bone marrow (Williams et al., 1990). In an initial attempt to evaluate the impact of G-CSF signaling in t(1;19) pre-B ALL cells we were unable to demonstrate a consistent proliferative response to G-CSF, either separate or in combination with several cytokines. This indicates that additional factors are required. In vivo, bone marrow stromal cells provide a variety of stimuli necessary for precursor B-cell maintenance and proliferation, like cytokines (including those used in this paper) and stimuli transduced through direct cellcell interactions (Bradstock and Gottlieb, 1995; Burrows and Cooper, 1993). However, it has proven dicult to reproducibly recapitulate the appropriate stromal environment in vitro. Speci®cally, the stimuli provided by direct cell-cell contact with stromal cells remain largely unresolved, although a number of adhesion molecules have been identi®ed (Bradstock et al., 1993). Further characterization of the stromal components involved in precursor B-cell development will be needed to address this point. Other groups have similarly attempted to identify target genes for E2A-Pbx1. They have made use of E2A-Pbx1-transfected 3T3 ®broblasts rather than preB cells (Fu and Kamps, 1997; McWhirter et al., 1997). Several genes were identi®ed which showed E2A-Pbx1dependent upregulation in 3T3 ®broblasts. However, these genes are either not expressed in t(1;19) cell lines (McWhirter et al., 1997), or no data have been reported concerning their expression in t(1;19) cell lines or tumor material (Fu and Kamps, 1997). The GCSFr was identi®ed using a pre-B cell line, and is also expressed in t(1;19) cell lines and tumor samples. In conclusion, our data support the hypothesis that the G-CSF reporter gene is one of the target genes for deregulation by E2A-Pbx1 in t(1;19) ALL. Further unraveling of the molecular pathways activated by the E2A-Pbx1 oncoprotein will shed light on the mechanisms underlying the development of t(1;19) pre-B ALL.

Materials and methods Plasmids pCDNA-E2A-Pbx1b was constructed by inserting the HindIII/EcoRI fragment from pJ3O-E2A-Pbx short (VanDijk et al., 1993) into the EcoRI/HindIII sites of pCDNAI/ amp (Invitrogen). pGK-Neo was a kind gift from Dr Hein ter Riele (Netherlands Cancer Institute, Amsterdam). Cell culture Reh, Sup-B27 and RCH-ACV were grown in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum, penicillin/streptomycin, 2 mM L-glutamine and 50 nM 2mercapto-ethanol. Reh-23s cells were grown in the same medium supplied with 1 mg/ml G418. Isolation of ALL cells from t(1;19) patients Following informed consent, pre-B ALL cells were isolated from the bone marrow of t(1;19) ALL patients by FicollHypaque (Nygaard, Oslo, Norway) density centrifugation.


T-cells were removed from the ALL cell samples by Erosetting with 2-amonoethylisothiouronium bromide (AET)-treated sheep red blood cells, followed by sedimentation through Ficoll-Hypaque (Madsen et al., 1980). Monocytes were removed by adherence to plastic petri dishes at 378C for 1 h. Transfections For the establishment of a cell line stably expressing E2APbx1 we used Reh cells (Rosenfeld et al., 1977). Cells were maintained at 556105 cells/ml. 56106 cells were resuspended together with 10 mg of pCDNA-E2A-Pbx1b and 1 mg pGK-Neo in 250 ml RPMI1640, and transfected by electroporation in a Biorad Gene-pulser at 250 V/ 960 mF. The electroporated cells were resuspended in 10 ml RPMI1640 with fetal calf serum and seeded in 96-wells plates at 5000 cells/well. Clones stably expressing E2APbx1b were isolated by selection with G418 (Boehringer, Mannheim) at 1 mg/ml and clonality was established by limiting dilution. This resulted in one clone (Reh-23s) which was used in the RDA procedure #1. For the RDA procedure #2, 56106 Reh cells were transfected as described above with either pCDNA-E2APbx1b (target) or the empty vector pCDNA1/amp (driver). RDA RDA was performed essentially as described (Hubank and Schatz, 1994), with primers as used in (Schutte et al., 1995). Oligonucleotides were obtained from Isogen (Maarsen, The Netherlands). The RDA products of round 3 were digested with DpnII to remove the adapter oligonucleotides, and subcloned into the BamHI site of pBluescript (Stratagene). The inserts were ampli®ed by PCR with T3 and T7 primers and sequenced on an ABI 373 stretch DNA sequencer using dye terminator chemistry (Applied Biosystems). The identity of the recovered RDA fragments was established using the BLAST algorithm (Gish et al., 1990). Analysis of G-CSFr expression To determine G-CSFr expression levels, cells were incubated at 48C for 60 min with biotinylated mouse anti-human G-CSFr monoclonal antibody LMM741 (10 mg/ml; Pharmingen, San Diego, CA). After washing, cells were treated at 48C for 60 min with 5 mg/ml of PEconjugated streptavidin. To further amplify G-CSFr signal, the cells were incubated 48C for 60 min with biotinylated mouse anti-streptavidin monoclonal antibody, and after washing, again with 5 mg/ml PE-conjugated streptavidin. During the ®nal incubation, FITC-conjugated mouse antiCD19 monoclonal antibody was included to stain all Blineage cells. Samples were analysed using a FACSCAN (Becton Dickinson, San Jose, CA).

Reverse transcriptase PCR Total RNA was prepared from 26106 log-fase cells according to Chomzynski and Sacchi (1987). DT16-primed ®rst-strand cDNA was prepared using AMV reverse transcriptase (Boehringer, Mannheim) in a 20 ml reaction according to the manufacturer's protocol. RT ± PCR was performed using the following primer pairs. b-actin primers: 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'CTCCTTAATGTCACGCACGATTTC-3' G-CSFr primers (Fukunaga et al., 1990): 5'-ACCTGGGCACAGCTGGAGTGG-3' and 5'-CAGGTCTCTGAGCTGTTATG-3'. 25 ml PCR reactions were performed using 0.5 ml of the ®rst-strand cDNA as template. Reactions contained 200 ng each of either G-CSFr- or b-actin-speci®c primers, 1 unit Amplitaq (Perkin-Elmer), 3 mM (G-CSFr) or 2 mM (b-actin) MgCl2 and 2.5 ml Perkin Elmer PCR buer II. Annealing temperatures were 668C for G-CSFr PCR and 528C for the b-actin PCR. Cycling times: (30''/948-30''/668 or 528-30''/728) for 25, 30 or 35 cycles. PCR products were analysed on a 2% agarose gel in the presence of 0.5 mg/ml ethidiumbromide. Products were visualized by UV and photographed. Analysis of proliferative response of primary t(1;19) pre-B ALL cells to G-CSF and additional cytokines DNA synthesis was assessed by uptake of [3H]thymidine [3H]TdR, speci®c activity 2 Ci/mmol, Amersham, UK). In brief, 0.26105 cells were cultured in triplicate in 96-wells dishes (Greiner, Alphen a/d Rijn, the Netherlands) for 3 and 7 days in 100 ml serum-free culture medium. Eighteen hours before harvesting on nitrocellulose ®lters using a Packard ®ltermate 196 (Packard Instrument Company, Meriden, CT), 0.1 mCi [3H]TdR was added to the cultures. Radioactivity was counted in a Topcount microplate scintillation counter (Packard). In addition to G-CSF (Amgen Inc., Thousand Oaks, CA) pre-B ALL cells were exposed to the following growth factors in this assay: IL-1 (Amgen Inc.), IL-2 (Cetus Inc.), IL-3 (Genetics Institute, Boston, MS), IL-4 (Immunex), IL-6 (Genetics Inst.), IL-7 (Immunex), FLT3 ligand, Stem cell factor and GM-CSF (Genetics Inst.). All factors were added to the cultures at optimal concentrations.

Acknowledgements The authors would like to thank Anita Schelen for expert technical assistance, and Dr M Schutte for expert advice on RDA. This work was supported by the Dutch Cancer Society (KWF) and NWO. MvD is supported by the Royal Dutch Academy for Arts and Sciences (KNAW).

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