M-CSF and M-CSF-receptor gene expression in acute myelomonocytic leukemias

Leukemia Research Vol, 14, No. 1, pp. 27-37, 1990. Printed in Great Britain.

(1145-2126/90 $3.00 + .00 Pergamon Press plc

M-CSF AND M-CSF-RECEPTOR GENE EXPRESSION IN A C U T E MYELOMONOCYTIC LEUKEMIAS M. R. PARWARESCH, H. KREIPE, J. FELGNER, K. HEIDORN, K. JAQUET, S. BODEWADT-RADZUNand H. J. RADZUN Institute of Pathology, Michaelisstr. 11, 2300 Kiel, Federal Republic of Germany

(Received 18 May 1989. Revision accepted 7 August 1989) Abstract--The role of hematopoietic growth factors in the pathogenesis of human leukemias is still obscure. In this study, RNA from 24 human acute myelomonocytic leukemias (AML) was used to analyze the expression of the macrophage colony stimulating factor (M-CSF) and its corresponding receptor (c-fms). Fifty percent of AML cells exhibited c-fms transcripts of regular length but at a lower level than in normal monocytes/macrophages. In most cases the reduced c-fms expression of AML cells was not associated with autostimulatory M-CSF expression. Only a few cases of AML showed co-expression of M-CSF and c-fins, which by contrast was regularly observed in cultivated blood monocytes and some tissue macrophage subsets. Higher levels of c-fms expression could be found in AMLs with a more mature monocytic immunophenotype. Permanent myelomonocytic cell lines expressed c-fms only after induction of monocytic differentiation. Neither the M-CSF gene nor the c-fms gene were rearranged in AML cells. In AML cells the homozygote genotype of the c-fms gene predominated. Our results do not provide evidence for the involvement of M-CSF and c-fins genes in human myeloid leukemogenesis, c-fms expression appears to indicate monocytic differentiation within the myelomonocytic lineage. We found autostimulatory M-CSF expression to be a physiologic feature of some tissue macrophages and hence not necessarily associated with neoplastic proliferation.

Key words: Autostimulation, c-fins, growth factors, M-CSF, myeiomonocytic leukemias.

INTRODUCTION

the other hand, CSFs cannot be regarded exclusively as growth promoting agents. They also induce differentiation and terminal maturation. These seemingly contradictory effects of CSFs result from a coupling of proliferation and differentiation in hematopoietic cell development [10, 11]. Consequently, whether CSFs play the role of a causative or antagonistic agents in the pathogenesis of A M L remains controversial [12-14]. In the present study we analyzed the expression of M-CSF and its corresponding receptor encoded by the proto-oncogene c-fms [15] in human A M L cells and found a reduced level of M-CSF receptor gene expression. Immunophenotypically more mature monocytic leukemias showed a higher level of c-fms expression, suggesting an association between c-fins expression and monocytic differentiation.

SELFRENEWAL,proliferation, differentiation and survival of hematopoietic cells are controlled by lineage specific growth factors or colony stimulating factors (CSFs) [1, 2]. It is not yet clear to what extent CSFs contribute to the escape of leukemic cells from external growth control. In murine leukemias growth factor and growth factor receptor genes have been revealed as target genes of viral insertional mutagenesis [3, 4]. Proliferation of acute myeloid leukemias (AML) can be enhanced in vitro by growth factors such as GM-CSF and interleukin 3 [5, 6] and growth factor receptors have been detected in the promyelocytic cell line HL-60 [7]. Furthermore, human A M L cells are capable of autocrine growth stimulation by simultaneous expression of growth factors and their corresponding receptors [8, 9]. On

Abbreviations: AML, acute myelomonocyticleukemias; CSF, colony stimulating factor; kb, kilo bases; M-CSF, macrophage colony stimulating factor; RFLP, restriction fragment length polymorphism; TPA, 12-0tetradecanoylphorbol 13-acetate. Correspondence to: H. Kreipe, Institute of Pathology, Michaelisstr. 11, 2300 Kiel, F.R.G.

MATERIALS AND METHODS

Leukemia samples Peripheral blood samples were collected from 24 patients with a diagnosis of acute non-lymphocytic leukemia (ANLL) based upon morphological and cytochemical criteria. Patients were included in the study when blasts 27

28

M.R. PARWARESCHet al.

exceeded 90% of mononuclear blood cells. The mononuclear blood cell fraction was separated by density gradient centrifugation and was used for analysis without additional purification. The morphological classification included French-American-British (FAB) classes 1 to 5 and a mean age of 58 years (median, 64; range 17-81).

Normal monocytes and tissue macrophages Peritoneal macrophages (PM; n = 4) were obtained from young sterile women undergoing diagnostic pelviscopy as described elsewhere [16]. Only PM samples with erythrocyte or granulocyte contamination lower than 1% as confirmed by Pappenheim staining of cytospin preparations were used (purity 97.7 -+ 1.5%). Alveolar macrophages (AM; n = 3; purity 98.3 --+0.5%) were harvested by bronchoalveolar lavage as described elsewhere [17]. Blood monocytes (BM) were isolated from peripheral blood of healthy volunteers (n = 5; purity 85.2 -+ 5%) and purified from the mononuclear blood cell fraction obtained after density gradient centrifugation (d = 1.077) by their ability to adhere to the bottom of the culture vessel during a 2 h incubation step at 37°C. Two BM samples were cultured in vitro for 48 h and one for 21 days in RPMI-1640 (Gibco, Heidelberg, F.R.G.) supplemented with 10% fetal calf serum (Boehringer, Mannheim, F.R.G.).

Leukemic cell lines U-937 cells [18], HL-60 cells [19] and THP-1 cells [20] were maintained in RPMI-1640 medium (Serva, Heidelberg, F.R.G.) supplemented with 10% fetal calf serum (Boehringer, Mannheim, F.R.G.). 12-O-tetradecanoylphorbol-13-acetate (Sigma, Munich, F.R.G.) induction was performed for periods of 24 and 48 h with 1.6 x 10 -8 M TPA as described elsewhere [21]. Retinoic acid treatment of HL-60 cells was performed for 6 days in a concentration of 1 x 10-7M (Sigma).

lmmunophenotyping of leukemic cells Cytocentrifuge preparations were used for immunostaining according to the method of Stein et al. (22) using peroxidase conjugated rabbit antimouse Ig diluted 1 : 10 in PBS (Dako, Copenhagen, Denmark) and supplemented with 2 vol.% heat-inactivated normal human serum and peroxidase conjugated goat antirabbit IgG diluted 1 : 10 in PBS (Medac, Hamburg, F.R.G.) as secondary and tertiary antisera, respectively. After visualization of peroxidase activity with 0.06% diaminobenzidine (Walter, Kiel, F.R.G.) and 0.01 vol.% H202 in PBS, preparations were counterstained with hematoxylin and mounted with glycerin gelatin. The following monoclonal antibodies were used: My7 (CD13; [23]), Tii9 (CD15, generously provided by Prof. Ziegler, Marburg, F.R.G.), VIM-2 [24], Ki-M1 (CDllc; [25]), Ki-M6 (CD68; I26]), and Ki-M8 [27].

Northern blot analysis Total cellular R N A was purified under ribonucleaseinactivating conditions using the guanidine thiocyanatecaesium chloride method [28] and analyzed by electrophoresis of 10/tg R N A through 1.2% agarose formaldehyde gels followed by Northern blot transfer to coated nylon membranes (Genescreen, New England Nuclear, Boston, U.S.A.) [29]. Immediately after measurement of R N A by spectralfluorometry (Kontron, Munich, F.R.G.), R N A from each sample was electrophoretically separated and blotted in order to allow for degradation-protected storage. The nylon sheets were then

cut into strips, each strip representing one electrophoretical lane. Strips from different samples were hybridized and exposed in common. The cloned v-fms gene pSM3 was the generous gift of Dr Sherr, Memphis, TN, U.S.A. [30]. A 1036 bp BglI/PstI fragment of the human M-CSF gene pcCSF-17 was obtained from Cetus Corporation, Emeryville, CA [31]. This fragment or the 1.5 kb PstI fragment of the pSM3 were isolated by preparative gel electrophoresis and labelled with 32P-deoxycytidinetriphosphate (3000 Ci/mmol; Amersham, Braunschweig, F.R.G.) by oligonucleotide priming (Multiprime, Amersham) to specific radioactivities of 1.5 x 109-3 × 109 c.p.m. p.g-i DNA. Membranes were prehybridized at 42°C for 24 h in buffer consisting of 50% formamide, 5 x SSC, 1% SDS, 2 x Denhardt's solution, 25 mM phosphate buffer (pH7.0), 5% dextran and 200~tgml -l salmon sperm DNA. The RNA blots were then hybridized for 24-48 h at 42°C with 1 × 107c.p.m. labelled DNA-probe per ml hybridization buffer (identical to the prehybridization buffer). After hybridization, the blots were washed twice in 2 x SSC and 0.5% SDS for 5 min at room temperature, twice in 0.2 x SSC, 0.5% SDS for 5 min at room temperature, and twice in 0.1 x SSC and 0.5% SDS for 30min at 55°C. After drying, blots were exposed to X ray films (Kodak X A R 5) with an intensifying screen at -70°C for one day or up to 20 days. The integrity of the RNA samples analyzed was checked by hybridization with the cloned actin gene obtained as a generous gift from Dr Kirschner, San Francisco, CA [32]. In each hybridization one or two samples were included, which had been already tested several times and from which it was known that they produced a signal of intermediate intensity. These samples served as a standard and the intensity of the hybridization signal of other samples was estimated by comparison and quantified by grading intensity between 1 (lowest) and 5 (highest). This was done in order to rule out differences due to different hybridization conditions and exposure times.

Southern blot analysis D N A obtained after density gradient centrifugation was digested with one of the three restriction enzymes BamHI, HindlII, or EcoRI (according to the manufacturers recommendations), separated by electrophoresis through 0.85% agarose gels and transferred to nylon membranes as described by Southern [33]. Prehybridization, hybridization, washings and exposure were done as described for Northern blots with slight modifications. Prehybridization lasted 2 h, washings were performed four times in 1 x SSC at room temperature and twice in the same buffer at 55°C for 15 min.

RESULTS c-fms and M - C S F gene expression in normal blood monocytes and tissue maerophages A s expected, c-fms transcripts o f a p p r o x i m a t e l y 4.2 kb could be detected in m o n o c y t e s (Fig. 1, lanes 1, 4). L o n g term cultivated m o n o c y t e s revealed a lower level of c-fins (Fig. 1, lane 7). W h e n the s a m e cell samples were hybridized with a r e c o m b i n a n t M - C S F gene p r o b e , little or no M - C S F R N A was d e m o n s t r a b l e in m o n o c y t e s cultivated for 48 h, but

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FIG. 1. During long term culture of monocytes two transcripts of the M-CSF gene are induced (lane 8) which can not be observed in freshly isolated or short term cultured monocytes (lane 2, 5). M-CSF expressing cultured monocytes show a relatively low level of c-fms gene expression (lane 7). Freshly isolated monocytes display a comparably low level of c-fms gene expression due to a transient downregulation during glass adherence; after 48 h of culture c-fms RNA levels increase (lane 4). Hybridization with an actin gene reveals that equal amounts of RNA have been blotted (lanes 3, 6, 9).

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FIG. 2. Normal alveolar macrophages show a 4.5 kb and a 3.3 kb M-CSF RNA transcript of the M-CSF gene (lane 2) and a relatively low level of c-fins gene expression (lane 1). Tissue macrophages of the peritoneal cavity reveal a high level of c-fins gene expression and no M-CSF RNA (lanes 4, 5). Hybridization with an actin gene reveals that equal amounts of RNA have been blotted (lanes 3, 6).

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not show c-fins expression (lanes 1, 7, 13). c-fms RNA is inducible in U-937 and HL-60 cells by TPA treatment for 48 h (lanes 4, 10). Transcripts of the M-CSF gene can not be detected in unstimulated and stimulated cell lines (lanes 2, 5, 8, 11, 14). Integrity of blotted RNA is shown by actin hybridization (lanes 3, 6, 9, 12, 15).

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31

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c-fins and M-CSFexpression in human AML monocytes cultivated for long terms exhibited two RNA variants of 4.5 kb and of 3.3 kb (Fig. 1, lanes 5, 8). These results emphasize an association of reduced c-fins R N A expression with an activation of M-CSF transcription in cultivated monocytes. Two tissue sites were used as sources of resident non-inflammatory macrophages. Alveolar and peritoneal macrophages revealed different levels of cfms expression (Fig. 2, lanes 1, 4). As in long-term cultured monocytes, the comparably low level of cfins expression in alveolar macrophages was associated with expression of M-CSF R N A with a size of 4.5 and 3.3 kb (Fig. 2, lane 2). c-fms and M-CSF gene expression in AML cells About 50% of the AML cell populations exhibited detectable levels of c-fms R N A expression when total cellular R N A was analyzed (Table 1). Most of the blast populations which stained positive for the mature monocytic markers Ki-M6 (CD 68) and KiM8 (unclustered) also expressed c-fms R N A (n = 11). Leukemic cells lacking the Ki-M6 and Ki-M8 antigens failed to exhibit detectable amounts of cfms R N A (n = 5). Out of 9 Ki-M8 negative samples one weakly expressed c-fins, while of 15 Ki-M8 positive samples four failed to express c-fins. All blast populations which showed more than 50% expression of the Ki-M8 antigen contained detectable amounts of c-fms RNA (n = 6). The amount of c-fins R N A demonstrable in leukemic cell samples was considerably lower than in normal monocytes or tissue macrophages (Table 1). Since analysis of non-neoplastic monocyte/macrophages revealed low levels of c-fms R N A when M-CSF R N A was present, we looked for M-CSF gene expression in AML cells as a possible cause for reduced growth factor receptor expression. Only four of the AML samples analyzed, however, exhibited M-CSF R N A (Table 1). In these cases a comparably high level of c-fins R N A was demonstrable (Table 1, No. 15, 16) or they did not express any c-fms R N A at all (Table 1; Fig. 3, lane 3). AML cells showed only a single size variant of M-CSF RNA of 4.5 kb. c-fins and M-CSF gene expression in myelomonocytic

cell lines Unstimulated U-937, THP-1 and HL-60 cells did not show any c-fms or M-CSF gene expression, c-fins expression was, however, inducible in both U-937 cells and HL-60 cells by TPA stimulation. After 48 h of stimulation a regular-sized transcript became detectable in both cell populations (Fig. 4). Retinoic acid failed to induce c-frns in HL-60 cells (data not shown). M-CSF R N A was also missing in TPA stimulated cell lines. Induction of c-fms R N A was

33

accompanied by expression of the Ki-M6 and partly by the Ki-M8 antigens (Table 2).

Structure of the c-fms and M-CSF gene in AML cells Southern blot analysis of the c-fms and M-CSF gene in AML cells and in cell lines U-937 and HL60 revealed no rearrangements or deletions (Fig. 5). AML No. 12 (Table 1) showed a restriction fragment length polymorphism of the M-CSF gene after EcoRI digest with an additional fragment of 9.0 kb which has already been described [34]. Since a restriction fragment length polymorphism of the c-fms gene defining two alleles has been described [35], we looked for the distribution of both alleles in AML cells. Most of the leukemic cell populations were homozygous for one allele (91.3%; 75% expected), whereas only two cases were heterozygous (8.6%; 23% expected). In no sample could homozygosity for the second allele (type aa; Xu et al., 1986) be detected. Cell lines U-937 and HL-60 were homozygous for the bb-type (data not shown). DISCUSSION Since their discovery and isolation, hematopoietic growth factors have been discussed as potential causative factors in leukemogenesis in animals as well as in man [1, 3, 5, 10, 34, 36, 37]. Among hematopoietic growth factors M-CSF/CSF-1 selectively stimulates the survival and growth of mononuclear phagocytes [38]. The exact role of M-CSF in human hematopoiesis in vivo is unclear. In vitro, however, it could be shown that human M-CSF induces BM to produce interferon and tumor necrosis factor, indicating a role in monocyte/macrophage activation [39]. In contrast to other CSFs the receptor of MCSF and its gene have been identified. It is encoded by the proto-oncogene c-fms [15], the cellular counterpart of the transforming retrovirus v-fins. The latter renders M-CSF dependent murine macrophages growth factor independent and tumorigenic by an unknown mechanism that differs from the physiologic mitogenic response to M-CSF [40, 41]. Autostimulatory M-CSF production in transfected macrophage cell lines is not sufficient to induce tumorigenicity [41]. Simultaneous expression of M-CSF and c-fms has been demonstrated in human AML cells and was interpreted as an autocrine escape from physiologic growth control [9]. Autostimulatory coexpression of M-CSF and c-fins by AML cells has also been described by others, but they found CSF1 expression to be associated with reduced growth capacity [34]. In our study M-CSF expression in AML cells was found in only four cases (Table 1). Possibly these divergent findings result from the different

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c-fins and M-CSF expression in human AML

TABLE 2. M-CSF AND c-fins EXPRESSION IN PROMYELOCYT1C AND HISTIOCYTIC CELL LINES

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purification procedures used for blasts, including short term culture. At least in monocytes, M-CSF RNA is rapidly inducible by adherence, although the M-CSF protein is not synthesized [42]. Interestingly, M-CSF R N A expression in A M L cells in these studies was not accompanied by functional activity [9, 34]. On the other hand, M-CSF can be detected in normal serum [43]. In addition, it remains questionable whether neoplastic cells have to produce their own growth factors when they are bathed in fluid containing CSF [2]. We found M-CSF gene expression to be the exception in neoplastic cells, whereas normal tissue macrophages of the lung and monocytes matured in vitro regularly showed co-expression of M-CSF and c-fins (Figs 1 and 2). Our results point to a role for M-CSF in normal macrophage maturation but not in neoplastic transformation. M-CSF has the capacity to antagonize neoplastic growth and to induce differentiation [34, 44, 45]. But there is considerable heterogeneity in the response of AML cells to growth factors, including M-CSF [6, 14, 46]. Therefore, we analyzed M-CSF receptor expression in AML cells in order to see whether variations occur between different AML types, cfms encoding the M-CSF receptor was only weakly expressed by AML cells as compared with monocytes (Fig. 3); in about 50% of cases it was completely missing. Normal precursor cells bear only few M-CSF receptors and their density increases with maturation [10]. Like precursor cells, leukemic cells have low receptor numbers for G-CSF and M-CSF [47]. The failure of some AML cells to express the receptor might explain their negative response to maturation inducing factors in vivo and in vitro. Whereas c-fins-negative A M L cells mostly lacked mature monocytic antigens, AML cells expressing cfins bore the Ki-M6 or Ki-M8 antigen (Table 1). Our results with permanent cell lines found that these monocytic antigens occurred simultaneously with cfins R N A after induction of maturation (Fig. 4, Table

2). Consequently, c-fms expression of AML cells can be considered as a marker of monocytic differentiation. Higher levels of c-fins expression in monocytic AML were also reported by Dubreuil et al. [48], who however detected fins transcripts in all FAB classification subtypes. In animals, involvement of the M-CSF gene and c-fins gene in leukemogenesis has been shown [4, 49]. This prompted us to look for structural changes of these genes in human AML cells. No rearrangements or deletions could be detected (Fig. 5). Our findings accord with those reported by others [34, 48]. The methods applied did not rule out point mutations within the c-fins gene, which have been shown to convert c-fms into a dominant cancer gene [50, 51]. The latter has been demonstrated, however, in mesenchymal non-hematopoietic cells [50, 51]. Lack of c-fins expression in permanent growth factor independent myelomonocytic cell lines (Fig. 4) and association of c-fins expression with monocytic differentiation (Table 1) argue against c-fins activation as an oncogene in these types of neoplasias. On the contrary, inactivation of one c-fins allele might contribute to leukemogenesis or at least lead to abnormalities in hematopoietic maturation, for example in 5q- syndrome [52]. Analysis of RFLP frequencies in AML cells showed that most AMLs are homozygous within the c-fins gene (Table 1). We are currently trying to establish T-cell lines from leukemic blood in order to see whether a loss of heterozygosity within the c-fins gene has occurred in leukemic clones. Furthermore, it remains to be shown whether point mutations of the c-fins gene will lead to pre-neoplastic or neoplastic abnormalities in hematopoietic maturation.

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c-fins and M-CSF expression in human AML

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39.

40.

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