Molecular cloning of the c-fms locus and its assignment to human chromosome 5

Molecular clones of the retroviral oncogene v-fms were used to isolate recombinant bacteriophages containing c-fms proto-oncogene sequences from a human placental DNA library. Viral and cellular fms sequences were used in Southern blotting experiments with a panel of 32 human X mouse somatic cell hybrids to assign the human c-fms proto-oncogene to human chromosome 5.     

Expression of the human c-fms proto-oncogene in hematopoietic cells and its deletion in the 5q- syndrome

The c-fms proto-oncogene was shown to be expressed in human bone marrow and in differentiated blood mononuclear cells, suggesting that its gene product plays a role in hematopoietic maturation. The c-fms mRNA was not detected in HL-60 cells, an established promyelocytic line, whereas c-fms expression appeared 48 hr after induction when most cells had differentiated into macrophages. An acquired deletion of chromosome 5 (5q-) in bone marrow cells is associated with abnormalities in blood cell production. The normal 5 and 5q- chromosomes were segregated by construction of cell hybrids between bone marrow and rodent cells. A selective system was used that requires retention of the structural gene for dihydrofolate reductase, located on human chromosome 5. Analysis of DNA from individual hybrid clones revealed that the 5q- deletion had removed the c-fms gene. We postulate that hemizygosity at the c-fms locus leads to abnormalities in hematopoietic maturation.     

Tandem linkage of human CSF-1 receptor (c-fms) and PDGF receptor genes

A 5' untranslated exon of the human CSF-1 receptor gene (c-fms) is separated by a 26 kb intron from the 32 kb receptor coding sequences. Nucleotide sequence analysis of cloned genomic DNA revealed that the 3' end of the PDGF receptor gene is located less than 0.5 kb upstream from this exon. Similarities in chromosomal localization, organization, and encoded amino acid sequences suggest that the genes encoding the CSF-1 and PDGF receptors arose through duplication. The as yet unidentified c-fms promoter/enhancer sequences may be confined to the nucleotides separating the two genes or could potentially lie within the PDGF receptor gene itself.     

Chromosomal localization of the human c-fms oncogene

A molecular probe was prepared with specificity for the human cellular homologue of transforming sequences represented within the McDonough strain of feline sarcoma virus (v-fms). By analysis of a series of mouse-human somatic cell hybrids containing variable complements of human chromosomes it was possible to assign this human oncogene, designated c-fms, to chromosome 5. Regional localization of c-fms to band q34 on chromosome 5 was accomplished by analysis of Chinese hamster-human cell hybrids containing as their only human components, terminal and interstitial deleted forms of chromosome 5. The localization of c-fms to chromosome 5 (q34) is of interest in view of reports of a specific, apparently interstitial, deletion involving approximately two thirds of the q arm of chromosome 5 in acute myelogenous leukemia cells.     

Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage.

The McDonough strain of feline sarcoma virus contains an oncogene called v-fms whose ultimate protein product (gp140v-fms) resembles a cell surface growth factor receptor. To identify and characterize the protein product of the proto-oncogene c-fms, antisera were prepared to the viral fms sequences and used to detect specific cross-reacting sequences in human choriocarcinoma cells (BeWo) known to express c-fms mRNA. Both tumor-bearing rat sera and a rabbit antiserum prepared to a segment of v-fms expressed in Escherichia coli detected a 140-kilodalton (kDa) glycoprotein in the BeWo cells. Tryptic fingerprint analysis of [35S]methionine-labeled proteins indicated that the viral fms proteins and the 140-kDa BeWo cell protein were highly related. This 140-kDa glycoprotein contained an associated tyrosine kinase activity in vitro and was labeled principally on serine after 32Pi metabolic labeling. These results suggest that the 140-kDa protein in BeWo cells is the protein product of the human c-fms proto-oncogene. This conclusion is supported by the finding that a similar protein is detectable only in other human cells that express c-fms mRNA. These other human cells include adherent monocytes and the cell line ML-1, which can be induced to differentiate along the monocyte-macrophage pathway. This is in agreement with current thought that the c-fms proto-oncogene product functions as the CSF-1 receptor specific to this pathway.     

A new acute transforming feline retrovirus with fms homology specifies a C-terminally truncated version of the c-fms protein that is different from SM-feline sarcoma virus v-fms protein.

The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of the SM-FeSV. The genome organization of the 8.6-kilobase HZ5-FeSV provirus is 5' delta gag-fms-delta pol-delta env 3'. The HZ5-and SM-FeSVs display indistinguishable in vitro transformation characteristics, and the structures of the gag-fms transforming genes in the two viruses are very similar. In the HZ5-FeSV and the SM-FeSV, identical c-fms and feline leukemia virus p10 sequences form the 5' gag-fms junction. With regard to v-fms the two viruses are homologous up to 11 amino acids before the C terminus of the SM-FeSV v-fms protein. In HZ5-FeSV a segment of 362 nucleotides then follows before the 3' recombination site with feline leukemia virus pol. The new 3' v-fms sequence encodes 27 amino acids before reaching a TGA termination signal. The relationship of this sequence with the recently characterized human c-fms sequence has been examined. The 3' HZ5-FeSV v-fms sequence is homologous with 3' c-fms sequences. A frameshift mutation (11-base-pair deletion) was found in the C-terminal fms coding sequence of the HZ5-FeSV. As a result, the HZ5-FeSV v-fms protein is predicted to be a C-terminally truncated version of c-fms. This frameshift mutation may determine the oncogenic properties of v-fms in the HZ5-FeSV.     

Lineage-specific expression of c-fos and c-fms in human hematopoietic cells: Discrepancies with the in vitro differentiation of leukemia cells

In vitro differentiation studies using the bipotential human leukemia cell line, HL60, have indicated that high levels of expression of two proto-oncogenes, c-fos and c-fms, are restricted to the myelomonocytic lineage. No such expression has been detected in induced granulocytic cells. In striking contrast to these observations, we found that c-fos mRNA levels are very high in purified human granulocytes, but barely detectable in blood monocytes and tissue macrophages. Human granulocytes contain, however, relatively low levels of c-fos protein, indicating that c-fos mRNA is inefficiently translated or that the protein is rapidly degraded in these cells. In closer agreement with the in vitro results, the level of the expression of c-fms is high in purified blood monocytes and undetectable in granulocytes. We found, however, that the evolution of monocytes into tissue macrophages is accompanied by a significant decrease in c-fms expression, suggesting that the function of c-fms is restricted to specific stages of monocytic differentiation. Our observations also show that results obtained using in vitro differentiation systems have to be regarded with caution, since they may not reflect the in vivo situation.     

Posttranscriptional suppression of proto-oncogene c-fms expression by vigilin in breast cancer

cis-acting elements found in 3'-untranslated regions (UTRs) are regulatory signals determining mRNA stability and translational efficiency. By binding a novel non-AU-rich 69-nucleotide (nt) c-fms 3' UTR sequence, we previously identified HuR as a promoter of c-fms proto-oncogene mRNA. We now identify the 69-nt c-fms mRNA 3' UTR sequence as a cellular vigilin target through which vigilin inhibits the expression of c-fms mRNA and protein. Altering association of either vigilin or HuR with c-fms mRNA in vivo reciprocally affected mRNA association with the other protein. Mechanistic studies show that vigilin decreased c-fms mRNA stability. Furthermore, vigilin inhibited c-fms translation. Vigilin suppresses while HuR encourages cellular motility and invasion of breast cancer cells. In summary, we identified a competition for binding the 69-nt sequence, through which vigilin and HuR exert opposing effects on c-fms expression, suggesting a role for vigilin in suppression of breast cancer progression.     

Induction of macrophage colony-stimulating factor-dependent growth and differentiation after introduction of the murine c-fms gene into FDC-P1 cells.

A system has been established for analyzing the functions of the c-fms/macrophage colony-stimulating factor (M-CSF) receptor gene product in hematopoietic growth and differentiation. The murine c-fms gene was introduced into the factor-dependent murine hematopoietic cell line FDC-P1 by retroviral infection, and conversion to M-CSF-dependent growth was assayed in agar cultures. Expression of the c-fms gene in FDC-P1 cells, which normally do not express this gene, resulted in the conversion of resultant FD(c-fms) cells to M-CSF-dependent growth. Stimulation of FD(c-fms) cells by M-CSF led to the formation of colonies of altered morphology and produced reversible morphological changes suggestive of myeloid differentiation. M-CSF also induced expression of mature myeloid surface marker proteins in the FD(c-fms) cells. Neither multi-CSF nor granulocyte-macrophage CSF induced similar phenotypic changes but remained able to stimulate the proliferation of undifferentiated FD(c-fms) cells. These results indicate that the c-fms gene was expressed functionally in FDC-P1 cells and transmitted signals for growth. Also, the interaction of M-CSF with the c-fms gene product generated an additional signal for myeloid differentiation but did not irreversibly commit FD(c-fms) cells to terminal differentiation. This system can be used for molecular analysis of the growth- and differentiation-promoting activities of the c-fms proto-oncogene.