M2 differentiation of MonoMac-1 cell line induced by M-CSF and glucocorticoid pathways.

Models of macrophage subtypes require molecular characterization to reliably facilitate their differentiation. Although CD16+ (Fc-gamma III receptor) monocytes that express CD163 (a hemoglobin/haptoglobin receptor) have been implicated in a variety of disease states, the conditions necessary to establish lineages of these cell subtypes remains unknown. The current investigations utilize a cell line derived from acute myelogenous leukemia lineage, MonoMac-1, to interrogate the factors that promote the development of CD16+ macrophages that express CD163. Results implicate the glucocorticoid pathway as well as c-fms signaling based on the action of dexamethasone and macrophage colony-stimulating factor-1 in promoting CD16+ expression, in addition to phorbol myristate acetate and lipopolysaccharides treatment. The ability of glucocorticoid and c-fms receptor antagonists to inhibit CD16+ cell formation further establishes the role of these pathways in CD16 expression in this cell line. In view of the inherent difficulty in working with primary cells as well as donor variation, cell lines may be preferable to primary cells for their consistency. We envision that the process we use to induce CD16 expression in this cell type will be useful for screening and identification of drug candidates potentially useful for the treatment of diseases where the etiology involves the expansion of the CD16+ monocytes subset or the accumulation of CD163+ tissue macrophages.

Induction of monocytic differentiation and NF-kappa B-like activities by human immunodeficiency virus 1 infection of myelomonoblastic cells.

The effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression.

A two-step, PU.1-dependent mechanism for developmentally regulated chromatin remodeling and transcription of the c-fms gene.

Hematopoietic stem cells and multipotent progenitors exhibit low-level transcription and partial chromatin reorganization of myeloid cell-specific genes including the c-fms (csf1R) locus. Expression of the c-fms gene is dependent on the Ets family transcription factor PU.1 and is upregulated during myeloid differentiation, enabling committed macrophage precursors to respond to colony-stimulating factor 1. To analyze molecular mechanisms underlying the transcriptional priming and developmental upregulation of the c-fms gene, we have utilized myeloid progenitors lacking the transcription factor PU.1. PU.1 can bind to sites in both the c-fms promoter and the c-fms intronic regulatory element (FIRE enhancer). Unlike wild-type progenitors, the PU.1(-/-) cells are unable to express c-fms or initiate macrophage differentiation. When PU.1 was reexpressed in mutant progenitors, the chromatin structure of the c-fms promoter was rapidly reorganized. In contrast, assembly of transcription factors at FIRE, acquisition of active histone marks, and high levels of c-fms transcription occurred with significantly slower kinetics. We demonstrate that the reason for this differential activation was that PU.1 was required to promote induction and binding of a secondary transcription factor, Egr-2, which is important for FIRE enhancer activity. These data suggest that the c-fms promoter is maintained in a primed state by PU.1 in progenitor cells and that at FIRE PU.1 functions with another transcription factor to direct full activation of the c-fms locus in differentiated myeloid cells. The two-step mechanism of developmental gene activation that we describe here may be utilized to regulate gene activity in a variety of developmental pathways.

Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations.

The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r-/- rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals.

E2a/Pbx1 oncogene inhibits terminal differentiation but not myeloid potential of pro-T cells.

E2a/Pbx1 is a fusion oncoprotein resulting from the t(1;19) translocation found in human pre-B acute lymphocytic leukemia and in a small number of acute T-lymphoid and myeloid leukemias. It was previously suggested that E2a/Pbx1 could cooperate with normal or oncogenic signaling pathways to immortalize myeloid and lymphoid progenitor cells. To address this question, we introduced the receptor of the macrophage-colony-stimulating factor (M-CSF-R) in pro-T cells immortalized by a conditional, estradiol-dependent, E2a/Pbx1-protein, and continuously proliferating in response to stem cell factor and interleukin-7. We asked whether M-CSF-R would be functional in an early T progenitor cell and influence the fate of E2a/Pbx1-immortalized cells. E2a-Pbx1 immortalized pro-T cells could proliferate and shifted from lymphoid to myeloid lineage after signaling through exogenously expressed M-CSF-R, irrespective of the presence of estradiol. However, terminal macrophage differentiation of the cells was obtained only when estradiol was withdrawn from cultures. This demonstrated that M-CSF-R is functional for proliferation and differentiation signaling in a T-lymphoid progenitor cell, which, in addition, unveiled myeloid potential of pro-T progenitors. Moreover, the block of differentiation induced by the E2a/Pbx1 oncogene could be modulated by hematopoietic cytokines such as M-CSF, suggesting plasticity of leukemic progenitor cells. Finally, additional experiments suggested that PU.1 and eight twenty-one transcriptional regulators might be implicated in the mechanisms of oncogenesis by E2a/Pbx1.

Induction of sustained expression of proto-oncogene c-fms by platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor, and its suppression by interferon-gamma and macrophage colony-stimulating factor in human aortic medial smooth muscle cells.

Vascular medial smooth muscle cells migrate, proliferate and transform to foam cells in the process of atherosclerosis. We have reported that the intimal smooth muscle cells express proto-oncogene c-fms, a characteristic gene of monocyte-macrophages, which is not normally expressed in medial smooth muscle cells. In the present study, we demonstrated that combinations of platelet-derived growth factor (PDGF)-BB and either epidermal growth factor (EGF) or fibroblast growth factor (FGF) induced high expression of c-fms in normal human medial smooth muscle cells to the level of intimal smooth muscle cells or monocyte-derived macrophages, whereas c-fms expression by PDGF-BB alone was 1/10 and both EGF and FGF had no independent effect on c-fms expression. By contrast, interferon (IFN)-gamma and macrophage colony-stimulating factor (M-CSF) suppressed the induction of c-fms expression. These results indicate that multiple growth factors and cytokines may play a role in the phenotypic transformation of medial smooth muscle cells to intimal smooth muscle cells in atherosclerotic lesions by altering c-fms expression.

Imbalanced gp130-dependent signaling in macrophages alters macrophage colony-stimulating factor responsiveness via regulation of c-fms expression.

The mechanisms by which interleukin-6 (IL-6) family cytokines, which utilize the common receptor signaling subunit gp130, influence monocyte/macrophage development remain unclear. Here we have utilized macrophages devoid of either gp130-dependent STAT1/3 (gp130(Delta STAT/Delta STAT)) or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase (gp130(Y757F/Y757F)) activation to assess the individual contribution of each pathway to macrophage formation. While the inhibition by IL-6 of macrophage colony-stimulating factor (M-CSF)-induced colony formation observed in gp130(wt/wt) mice was abolished in gp130(Delta STAT/Delta STAT) mice, inhibition of macrophage colony formation was enhanced in gp130(Y757F/Y757F) mice. In gp130(Delta STAT/Delta STAT) bone marrow-derived macrophages (BMMs), both IL-6- and M-CSF-induced ERK1/2 tyrosine phosphorylation was enhanced. By contrast, tyrosine phosphorylation of ERK1/2 in response to M-CSF was reduced in gp130(Y757F/Y757F) BMMs, and the pattern of ERK1/2 activation in gp130 mutant BMMs correlated with their opposing responsiveness to M-CSF-induced proliferation. When compared to the level of expression in gp130(wt/wt) BMMs, c-fms expression was elevated in gp130(Delta STAT/Delta STAT) BMMs but reduced in gp130(Y757F/Y757F) BMMs. Finally, an ERK1/2 inhibitor suppressed M-CSF-induced BMM proliferation, and this result corresponded to a reduction in c-fms expression. Collectively, these results provide a functional and causal correlation between gp130-dependent ERK MAP kinase signaling and c-fms gene activation, a finding that provides a potential mechanism underlying the inhibition of M-CSF-dependent macrophage development by IL-6 family cytokines in mice.

Lack of TP53 and FMS gene mutations in children with myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are rare disorders in children. Molecular mechanisms underlying MDS in children are not yet completely understood. Considering the role of FMS and TP53 gene mutations in adult MDS patients, we analyzed mutations of these genes in a cohort of 35 children with MDS. Single-strand conformation polymorphism polymerase chain reaction analysis performed on FMS codon 969 and TP53 exons 5-9 showed no mutations in the analyzed sequences. Our results suggest that molecular mechanisms of MDS evolution in children are different from those in adults.

DNA repair in the MYC and FMS proto-oncogenes in ultraviolet light-irradiated human HL60 promyelocytic cells during differentiation.

In order to better understand the role of transcription in cellular processing of damage in specific DNA sequences, we have used an in vitro differentiation system to modulate the activity of the MYC gene. When human HL60 promyelocytic cells differentiate in vitro, the transcriptional activity of the MYC gene is down-regulated. We have shown that in the expressed MYC gene, 56% of UV-induced cyclobutane pyrimidine dimers (CPDs) are removed within 18 h and the transcribed strand is selectively repaired. However, late in differentiation, when the MYC gene is maximally down-regulated, only 15% of the CPDs are removed within the same period. During early differentiation, the MYC gene is regulated by a block to transcription elongation at the 5' end of the first intron. Our results reveal no significant difference in the rate of CPD removal between the restriction fragments upstream and downstream of this elongation block. Furthermore, both strands of each fragment exhibit similar repair characteristics. In contrast, the constitutively expressed FMS gene exhibits proficient removal of CPD in both the differentiated and undifferentiated cells. Furthermore, the repair appears to be more proficient at the 5' end (exon 1) than in the 3' end of the gene about 35 kilobases downstream from exon 1. Since efficient repair of the active FMS gene is maintained in the differentiated cells the loss of repair competence seen in MYC is more likely associated with its reduced transcriptional activity than with a decrease in the overall repair capacity of the terminally differentiated cells.

Cloning and structural analysis of the human c-kit gene.

The recent identification of the mouse White spotting and Steel loci as genes encoding the c-kit receptor and its ligand, respectively, has shed light on the importance of this ligand and receptor in embryogenesis, melanogenesis and hematopoiesis. In order to determine if the c-kit proto-oncogene is involved in human disease, we isolated seven overlapping lambda recombinants, using a fetal brain cDNA, and characterized the normal human gene (KIT). The longest mapped transcript is 5230 bp, is alternatively spliced and includes 21 exons that span more than 70 kb of DNA. From the exon-intron structure, we have localized an alternative splice site to the 3' end of exon 9. The overall c-kit gene structure closely resembles that found in the CSF-1R gene (c-fms). This similarity includes a large first intron, the same number of exons containing translated sequence and very similar exon-intron boundaries. Using pulsed-field gel electrophoresis, we have linked KIT to the platelet-derived growth factor receptor A gene, with both residing on a 700-kb BssHI fragment. These data will allow investigation into the control of KIT expression and the potential to identify mutations or altered expression of this gene in human disease.

Two new polymorphisms but no mutations of the KIT gene in patients with myelodysplasia at positions corresponding to human FMS and murine W locus mutational hot spots.

The KIT proto-oncogene encodes a tyrosine kinase receptor which plays a critical role in haemopoiesis. We have screened genomic DNA from bone marrow mononuclear cells of 46 patients with myelodysplasia (MDS) for mutations/deletions of exons 6, 13, 17, and 21 of the KIT gene (stem cell factor receptor) using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and autoradiography to detect single-stranded conformational polymorphisms (SSCP). These exons include positions analogous to those mutated in the FMS gene (colony-stimulating factor-1 receptor) in myelodysplastic syndrome (MDS) and mutated/deleted in the Dominant White Spotting mouse (W locus) which results in macrocytic anaemia. Two different gel running conditions were used for each exon. Polymorphisms were identified only at 4 degrees C in exon 17 (three out of 44 MDS samples and two of 21 DNA samples from normal subjects), and in the non-coding region of exon 21 (five out of 34 MDS samples and seven out of 19 normals). Direct sequencing identified a G to A base change at nucleotide 3169 within exon 21, and a C to T change at position 2415 in exon 17. No conformational changes suggestive of mutations or deletions have been found to date, although we cannot rule out low frequency clonal abnormalities undetectable by our method, which has a sensitivity in our hands of approximately 5%. Polymorphisms occur frequently in the KIT gene. Together with this study, a total of five have been described.

All-trans retinoic acid induces monocyte growth factor receptor (c-fms) gene expression in HL-60 leukemia cells.

All-trans-retinoic (ATRA) treatment of patients with acute promyelocytic leukemia results in differentiation of the malignant cells and a high complete remission rate. ATRA treatment induced granulocytic differentiation in HL-60 cells as assessed by nitroblue tetrazolium (NBT) reduction, but had no effect on non-specific esterase (NSE) straining, as expected in cells maturing along the monocytic lineage. However, our results demonstrate that ATRA (0.1-10 microM) induces expression of the c-fms (monocyte colony-stimulating factor receptor) gene in HL-60 cells. This effect was detectable after 2 days and expression was maximal at 5 days. Similar results were obtained during treatment with cis-retinoic acid (CRA), hexamethylene bisacetamide (HMBA), or dimethyl sulfoxide (DMSO). The results also demonstrate that ATRA-induced c-fms expression is potentiated by exposure to tumor necrosis factor alpha (TNF alpha) or dibutyryl cyclic adenosine monophosphate (cAMP). The induction of c-fms transcripts by ATRA is associated with induction of M-CSF-binding ability, suggesting cell surface expression of the monocyte growth factor receptor. Our results indicate that retinoic acid can induce features of both monocytic and granulocytic differentiation in HL-60 cells.

Synergy between SCF or M-CSF with IL-3 or GM-CSF in FDC-P1 cells: a sensitive assay of transforming mutations of c-fms.

Stem cell factor (SCF) was found to stimulate the growth of the haemopoietic cell line FDC-P1 in synergy with either interleukin 3 (IL-3) or granulocyte-macrophage-colony stimulating factor (GM-CSF). Similarly, macrophage colony-stimulating factor (M-CSF) was shown to synergize with IL-3 or GM-CSF, following the infection of FDC-P1 cells with a recombinant retrovirus which encoded the receptor for M-CSF (M-CSFr). These results raise the possibility that signal transduction pathways which are controlled by SCF in FDC-P1 cells, can be activated by M-CSF if its receptor is illicitly expressed. FDC-P1 cells that expressed the M-CSFr were responsive to as little as 100 U/ml of M-CSF when added in combination with IL-3 or GM-CSF. This sensitive assay was used to demonstrate that transforming deletions of the C-terminal tail of the M-CSFr and two-point mutations within the same region that converted tyrosine 969 to either phenylalanine or to cysteine, allowed the mutant M-CSF receptors to synergize with IL-3 or GM-CSF in the absence of M-CSF. These mutations were found to be more evidently transforming in FDC-P1 cells than in Rat-2 fibroblasts. The possible relevance of these results to leukaemia and to gynaecological malignancies is discussed.

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression.

The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene.

Repression of the c-fms gene in fibroblast cells by c-Myc-MM-1-TIF1beta complex.

MM-1 has been reported to repress the E-box-dependent transcription activity of c-Myc by recruiting histone deacetylase 1 complex via TIF1beta/KAP1. In this study, to identify target genes for c-Myc-MM-1-TIF1beta, we established rat-1 cells harboring the dominant-negative form of TIF1beta to abrogate the pathway from TIF1beta to MM-1-c-Myc. This cell line, in which transcription activity of c-Myc was activated, was found to be tumorigenic. By DNA-microarray analysis of this cell line, expression and promoter activity of the c-fms oncogene were found to be upregulated. Of the two promoters, pE1 and pE2, in the c-fms gene, pE1 promoter activity was found to be activated in an E-box-dependent manner.

Gene mutations in myelodysplasia.

The myelodysplastic syndrome is a paradigm of human preleukaemia. Normal haemopoiesis is progressively displaced by an abnormal clone derived from a mutated stem cell. The initial mutation is unknown but its occurrence may be related to the overall load of random mutations which are a consequence of both intrinsic DNA defects and external mutagens. Evolution of the pathological population is marked by an increasing load of genetic lesions at the molecular and cytogenetic levels. Ras mutations can be detected in the blood of about 50% of MDS patients. Fms mutations are less common but these lesions can be found both in patients and in haematologically normal subjects who have previously received cytotoxic therapy suggesting that they can occur early in the preleukaemic process. Clonal haemopoiesis in the absence of either ras or fms mutations can occur in these subjects. The data suggest the inability of mutant ras or fms genes alone to produce observable preleukaemic changes but that subjects with these mutations may be predisposed to future MDS. Ras mutations are a common accompaniment of a wide variety of malignancies and experimental transfection of the mutant gene can induce a malignant phenotype in cultured cells. There are many possible mechanisms for this transformation which may be relevant in a clinical context. Experimentally observed effects include a direct influence on the cell cycle, the induction of drug resistance and the stimulation of autocrine growth factor production. It may eventually be possible to define which gene mutations are important in conferring a malignant state, which determine phenotype and which are of incidental significance.

Biological consequences of a point mutation at codon 969 of the FMS gene.

The FMS proto-oncogene encodes the cell surface receptor for colony stimulating factor-1 (CSF-1). Mutations of the FMS gene at codon 969, in the C-terminal region of the gene, have been detected in haematological malignancies. To ascertain the biological significance of a mutation at this codon, we have used a murine haematopoietic cell line, FDC-P1, containing a mutation at codon 969 that results in a phenylalanine replacing a tyrosine. FMS 969 mutant cells and v-fms transfected cells conferred interleukin 3 (IL-3) independent stimulation of FDC-P1 cells, whereas cells transfected with a wild-type FMS construct required exogenous IL-3 for growth. FDC-P1 cells containing a FMS 969 mutation and v-fms transfected cells were tumorigenic in nude mice. Binding studies with radioidonated CSF-1 revealed saturable specific binding in FMS wild-type cells with a Km of 0.9 mM; however, mutant FMS-containing cells did not display saturation kinetics, but instead exhibited a linear relationship between ligand concentration and amount bound. Constitutive expression of FOS was detected in 969 mutant cells in the absence of exogenous CSF-1, a phenotype that was only inducible in wild-type cells in response to CSF-1. FOS and JUNB expression by v-FMS transfected cells showed a similar pattern to FMS wild-type cells. This mutation has been detected in patients with haematological malignancies, and illustrates that the pathway of FMS 969 phenylalanine mutations and v-fms induced pathogenesis can be distinguished. These data indicate that there is a biological role for FMS codon 969 phenylalanine mutation which results in transformation of FDC-P1 cells.

The promoter of macrophage colony-stimulating factor receptor is active in astrocytes.

Macrophage colony-stimulating factor (M-CSF) is a hematopoietin whose actions are essential for growth and survival of macrophages, placental development, ramification of microglia and tumor progression. The expression of the receptor for macrophage colony-stimulating factor (c-fms) is regulated by two distinct promoters: distal and proximal. The distal promoter is active in trophoblasts during embryogenesis and the proximal promoter directs expression to the cells of myeloid lineage. Here we report the generation of transgenic mice expressing beta-galactosidase under the control of the human proximal c-fms promoter and demonstrate the promoter activity in astrocytes, cells of neurological origin that partially take over the role of the macrophages in the central nervous system. Enzymatic activity of beta-galactosidase was detected in homogenated spleen, bone marrow and brain and in the cell extracts from peritoneal macrophages of transgenic mice. Immunohistochemical staining of brain showed the presence of beta-galactosidase in astrocytes. We hypothesize that M-CSF released by astrocytes, upon stimulation by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) or interleukin-1 (IL-1), regulates the expression of its own receptor.

The role of CSF-1 in normal physiology of mammary gland and breast cancer: an update.

Colony stimulating factor (CSF-1) and its receptor (CSF-1R, product of c-fms proto-oncogene) were initially implicated as essential for normal monocyte development as well as for trophoblastic implantation. However, studies have demonstrated that CSF-1 and CSF-1R have additional roles in mammary gland development during pregnancy and lactation. This apparent role for CSF-1/CSF-1R in normal mammary gland development is very intriguing because this receptor/ligand pair has also been found to be important in the biology of breast cancer in which abnormal expression of CSF-1 and its receptor correlates with tumor cell invasiveness and adverse clinical prognosis. Recent findings also implicate tumor-produced CSF-1 in promotion of bone metastasis in breast cancer, and a certain membrane-associated form of CSF-1 appears to induce immunity against tumors. This review aims to summarize recent findings on the role of CSF-1 and its receptor in normal and neoplastic mammary development that may elucidate potential relationships of growth factor-induced biological changes in the breast during pregnancy and tumor progression.

Sporadic amplification of the c-fms proto-oncogene in human musculoskeletal sarcomas.

Fresh samples of bone and soft tissue sarcoma from 31 patients were analyzed by the Southern technique for amplification or other structural abnormalities of the c-fms oncogene. As a sole finding, amplification of the c-fms (4- and 10-fold) was detected in 2 of 3 cases with a histologic diagnosis of liposarcoma. RFLP analysis disclosed no gene rearrangements associated with the c-fms amplification. The case with the highest amplification of the c-fms gene was disease-free at review. The 2 fms-amplified liposarcomas were aneuploid according to DNA flow cytometry. This is the first study demonstrating amplification of the c-fms proto-oncogene in human liposarcoma. The fact that this structural abnormality of the c-fms gene was only detected in liposarcomas might point to an uncommon but tissue-specific phenomenon.