Proc. Nati. Acad. Sci. USA Vol. 84, pp. 4480-4484, July 1987 Cell Biology Differential expression and regulation of the c-src and c-fgr protooncogenes in myelomonocytic cells (bone marrow-derived macrophages/protein tyrosine kinases/myeloid leukemia/colony-stimulating factors) CHERYL L. WILLMAN*t, CARLETON C. STEWARTt, JEFFREY K. GRIFFITH*, SIGRID J. STEWARTt, AND THOMAS B. ToMASI*§¶ Departments of *Cell Biology and tPathology, University of New Mexico School of Medicine, Albuquerque, NM 87131; and *Experimental Pathology Group, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545 Communicated by H. Sherwood Lawrence, February 20, 1987 (received for review August 21, 1986)

ABSTRACT To study the expression of src-related proto- while all of these genes encode proteins with highly homol- oncogenes during the development of myeloid cells and the ogous carboxyl-terminal kinase domains, their amino-termi- regulation of these genes by the colony-stimulating factors that nal domains are essentially unrelated. This amino-terminal control myelopoiesis, normal monocytic cells at distinct stages diversity may allow each gene to be independently regulated of differentiation were derived from murine bone marrow with and expressed in distinct cell lineages and may determine the the monocytic lineage colony-stimulating factor CSF-1. substrate specificity, intracellular location, and unique func- Protooncogene expression was also examined in uncultured tions of the encoded proteins. human myeloid leukemia cells. While c-src transcripts were In several reports (19-22), myelomonocytic cells have detected in myeloid leukemia cells representative of all stages been rendered growth factor independent by infection with of differentiation, the highly related gene c-fgr was expressed retroviruses containing oncogenes of the src family. In each at high levels only at later developmental stages, both in normal case, the viral oncogene product was capable of triggering cells committed to the monocytic lineage and in leukemic cells proliferation normally controlled by the binding of a hema- with a differentiated myelomonocytic phenotype. When bone topoietic growth factor to its receptor. Because the expres- marrow-derived monocytic cells were synchronized and stim- sion of v-src-related genes was linked to the control of ulated to proliferate with CSF-1, c-fgr transcripts (but not hematopoiesis, we were interested in determining whether transcripts from the highly related genes c-src or c-yes) were src-related protooncogenes were differentially expressed in induced 8 hr after the addition of CSF-1 and decreased to low normal hematopoietic cells and whether the expression of levels by 20 hr as the monocytic cells entered S phase. The these genes could be modulated by hematopoietic growth selective induction of c-fgr mRNA by CSF-1 suggests that this factors [termed colony-stimulating factors (CSFs)], which tyrosine kinase may have a unique function in normal control proliferation and differentiation in the myeloid lin- monocytic cells, distinct from other src-related tyrosine eage (reviewed in ref. 23). kinases. MATERIALS AND METHODS Phosphorylation of proteins on critical tyrosine residues has been linked to the control of proliferation in both normal and In Vitro Bone Marrow Culture System and Cell Culture transformed cells (1). Tyrosine protein kinase activity is an Conditions. Bone marrow cells were obtained from the intrinsic property of the transforming proteins of certain femurs of C3H/Anf mice (4-8 weeks old) by flushing the oncogenic retroviruses. In addition, tyrosine kinase activity marrow cavity with 3 ml of a-MEM (GIBCO) supplemented is a hallmark of at least four polypeptide growth factor with glutamine (2 mM), sodium bicarbonate (0.38%), 10% receptors that are related to the pp60v-src transforming protein heat-inactivated fetal bovine serum (HyClone, Logan, UT), of Rous sarcoma virus in their carboxyl-terminal kinase penicillin/streptomycin (100 units/ml), and 10% L929 cell- domains: the insulin and platelet-derived growth-factor re- conditioned medium. Cells were cultured at 37°C with 10% ceptors, the monocyte lineage-specific colony-stimulating C02/90% air and high humidity for 24 hr to remove adherent factor (CSF-1) receptor (encoded by c-fms), and the epider- stromal cells; the nonadherent cells were then collected, mal adjusted to a concentration of 2 x 104 nucleated cells per ml growth-factor receptor (encoded by c-erbB) (1-3). in the same liquid growth medium, and 30 ml was plated per Another set of pp60v-src-related cellular proteins with 75-cm2 flask. After 6 days, the cultures produced highly intrinsic tyrosine kinase activity (encoded by c-src, c-fgr, enriched populations of nonadherent and adherent mono- c-yes, c-syn, c-lyn, c-abl, c-fps/fes, and Ick) do not possess cytic cells (24, 25). the transmembrane protein structures characteristic of Cell-Cycle Analysis, Monoclonal Antibody Staining, and growth-factor receptors, and their functional roles in normal Flow Cytometric Techniques. The panel of rat anti-mouse cells remain elusive. The best characterized protein of this primary monoclonal antibodies to hematopoietic cell-surface group is pp60csrc, a membrane-associated protein, which has antigens described in this study, as well as our methods for been reported to be involved in both the transduction of monoclonal antibody staining, flow cytometric analysis, growth-controlling signals (4-6) and in specialized functions cell-cycle analysis, and cell sorting have been described (24, in fully differentiated nonproliferating cells (7-10). The con- cell- served intron-exon structures of c-src (11, 12), c-fgr (11, 13, 26). Analysis of antibody binding to human myeloid 14), c-yes (14, 15), c-syn (16), c-lyn (17), and Ick (18) suggest surface antigens was detected by flow cytometric techniques that these genes arose by duplication from an ancestral c-src using the monoclonal antibodies anti-HLA-Dr (I2); My4, gene. Preliminary sequence information has determined that, Abbreviations: CSF, colony-stimulating factor; CSF-1, monocyte lineage-specific CSF. The publication costs of this article were defrayed in part by page charge §Present address: Roswell Park Memorial Institute, 666 Elm Street, payment. This article must therefore be hereby marked "advertisement" Buffalo, NY 14263. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed. Downloaded by guest on September 23, 2021 4480 Cell Biology: Willman et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4481 My7, and My9 (Coulter); and MylO (HPCA-1) (Becton- Volume Dickinson). RNA Isolation and Hybridization Analysis. Total RNA was isolated by lysing cells in 4 M guanidinium thiocyanate. Purification of total RNA was completed by ultracentrifuga- tion of the guanidinium lysate over cesium chloride as described (27). For RNA blot analysis, 10 ,ug oftotal RNA per sample was electrophoresed through 1% agarose-formalde- hyde denaturing gels, transferred to nitrocellulose (28), and Ca hybridized as described (28, 29). RNA blots were exposed to film with screens for 24-48 hr at -70'C. x-ray intensifying 0 Quantitation of the extent ofhybridization was performed by 6 soft laser scanning densitometry of autoradiographs. c Cloned DNA fragments, purified from vector sequences by preparative agarose gel electrophoresis, were nick-translated Ca in the presence of [32P]dCTP. Specific fragments used in this study included the following: v-src, 0.8-kilobase (kb) Pvu II fragment (30); c-src, 0.6-kb BamHI fragment from human exons 11 and 12 (12); pv-fgrl, 0.8-kb Ava I/BamHI fragment (13); yes, 1.15-kb Pst I fragment from nucleotides 1652-2750 (15); v-fms, 1.3-kb Pst I fragment (31); v-fos, 1.0-kb Pst I fragment (32); v-myc, 2.9-kb BamHI fragment (33); and pGAD-28 (glyceraldehyde-3-phosphate dehydrogenase), 1.2- kb Pst I fragment (34). 128 RESULTS Channel FIG. 1. Expansion of the monocytic lineage from in vitro bone Characterization of Normal Monocytic Populations Derived marrow culture. Cell lineages present per day ofin vitro bone marrow from in Vitro Bone Marrow Cultures. Murine monocytic cell culture in the presence ofCSF-1 were assessed by analyzing the cell populations at distinct stages of differentiation were derived volume profiles ofthe cultures in a fluorescence-activated cell sorter. from short-term in vitro culture of bone marrow precursor Cells from each volume peak were sorted and analyzed cytochemi- cells in medium containing fetal bovine serum and the cally and morphologically (24). The predominant cell population monocytic colony-stimulating factor CSF-1, derived from contained within each volume peak per day is designated in the L929 cell-conditioned medium (see Materials and Methods; figure. Day-6 cultures were composed predominantly of monocytic refs. 24, 25, and 35). Initial cultures containing heterogenous cells with two distinct volume distributions: 10% of the total cells in bone marrow cells resolved two the culture were small nonadherent cells (90%6 of which were MAC (Fig. 1, day 0) into popula- 1+ and nonspecific esterase-positive), while >90%o of the cells in tions of highly enriched monocytic cells by day 6 (Fig. 1, day culture were adherent differentiated (MAC1+, -2+, -3+) monocytes- 6): a population of small immature nonadherent monocytic macrophages. cells that stained with the MAC 1 monoclonal antibody (detecting the C3bi complement receptor) and a population of large MAC 1, MAC 2, MAC 3-positive adherent monocytes- 10-fold higher levels of a src-related mRNA [detected by macrophages (24). The bone marrow cells of other lineages dot-blot hybridization with a v-src probe (30)] and 20-fold die in culture in the absence of their specific hematopoietic higher levels of c-fos mRNA relative to the adherent mac- growth factors. rophages (data not shown). RNA blot hybridization revealed Progressive differentiation in the hematopoietic cell lin- that the src-related gene expressed at high levels in the bone eages is associated with decreasing proliferation. To deter- marrow-derived monocytic cells was c-fgr. Initial hybridiza- mine the extent ofproliferation of each ofthe two monocytic tion of the day-6 cells with a v-src probe [capable of populations, we measured the DNA content per cell by flow hybridizing with transcripts from either c-src or c-fgr (11)] cytometric techniques (26). Day-6 nonadherent cells had a detected only a 2.6-kb mRNA transcript in the murine DNA content distribution in which 63% ofthe total cells were monocytic cells (data not shown). Hybridization with the in the Go/Gj phase of the cell cycle with 2n DNA content, fgr-specific probe under stringent conditions revealed the 26% of the cells were in S phase with DNA contents varying same 2.6-kb band in nonadherent cells (Fig. 2A, lanes 1 and from 2n to 4n, and 12% were in G2/M with 4n DNA content 2). With differentiation to adherent macrophages, the level of (data not shown). Day-6 adherent monocytes had 90%6 ofcells the 2.6-kb c-fgr transcript decreased by 90% (Fig. 2A, lanes with 2n DNA content in Go/Gj, 6% of cells with 2n-4n DNA 3 and 4). Hybridization with c-src and yes-specific probes content in S phase, and 4% of cells with 4n DNA content in G2/M. Since the cell-cycle transit time of both the nonad- A B C D herent and adherent monocytic cells was essentially equiv- 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 alent (=16 hr; unpublished observations), these cell-cycle ii ^ ~~~~~4.7-W ww distributions implied that the day-6 nonadherent monocytes 2 6- 1 - (with 38% of cells in S + G2/M) had a 4-fold greater fraction 2.2- 00 1.3- of proliferating cells when compared to day-6 adherent cells (with <10% of cells in S + G2/M). c -fgr C- fos C-fins gpd Thus, these short-term in vitro bone marrow cultures yield two of cells at distinct of FIG. 2. RNA blot hybridization of bone marrow-derived mono- populations monocytic stages cytic cells. Ten micrograms of total RNA from independent isolates differentiation: immature nonadherent proliferating mono- ofday-6 nonadherent monocytic cells (lanes 1 and 2) and independent cytes and more highly differentiated monocytes-macro- isolates of day-6 adherent cells (lanes 3 and 4) were hybridized with phages, which have essentially ceased to proliferate. probes specific forfgr (A), c-fos (B), c-fms (C), and the constitutively Protooncogene Expression In Developing Monocytic Cells. expressed gene glyceraldehyde-3-phosphate dehydrogenase (gpd) Day-6 nonadherent monocytic cells were found to contain (D). Downloaded by guest on September 23, 2021 4482 Cell Biology: Willman et al. Proc. Natl. Acad. Sci. USA 84 (1987) revealed no detectable transcripts at either stage of differen- bovine serum. At 24 hr (time 0), the quiescent cells were tiation under high or low stringency conditions (data not washed and stimulated to proliferate by the addition of fresh shown). RNA blot analysis also revealed high steady-state medium supplemented with 10% fetal bovine serum and levels of the 2.2-kb c-fos transcript in the nonadherent either 10% L929 cell-conditioned medium containing CSF-1 monocytes (Fig. 2B, lanes 1 and 2), which decreased to very or stage IV-purified CSF-1 (2000 units/ml) (from L929 low levels with differentiation to the relatively quiescent cell-conditioned medium, generously provided by Hsiu-san adherent cells (Fig. 2B, lanes 3 and 4). As shown in Fig. 2C, Lin, Washington University, St. Louis). Total RNA was the 4.7-kb c-fms mRNA transcript [encoding the CSF-1 isolated from these cells at various lengths of time after they receptor (2, 3)] was expressed in both populations, although were stimulated to proliferate with CSF-1, and the patterns of independent isolates of day-6 adherent cells consistently gene expression were compared in RNA blot analysis (Fig. 3 contained 1.5-2 times the c-fms mRNA level detected in the B and C). [3H]Thymidine uptake was used to simultaneously nonadherent cells (Fig. 2C). In contrast to the differential monitor the entry of cells into S phase (Fig. 3A) (35). expression of c-fgr and c-fos, the constitutive gene glycer- Stimulation ofthe quiescent cells with CSF-1 led to a rapid, aldehyde-3-phosphate dehydrogenase [gpd (34)] was ex- transient, 40-fold induction of c-fos mRNA within 30 min of pressed at similar levels in all isolates of day-6 nonadherent exposure of the cells to the growth factor, decreasing to and adherent cells (Fig. 2D). baseline levels by 2 hr (Fig. 3). In addition to this rapid first Induction ofc-fgrExpression in Monocytic Cells with CSF-1. phase of c-fos mRNA induction, a second phase of c-fos Since the day-6 monocytic populations were at distinct expression (representing a 10- to 15-fold induction from developmental stages, the differences in the steady-state baseline levels) began at 4 hr and persisted at relatively levels of c-fgr and c-fos mRNA in the nonadherent and more constant levels thereafter (Fig. 3). Levels of c-myc mRNA highly differentiated adherent monocytic cells could be were induced 2-4 hr after the readdition ofthe CSF-1 growth interpreted as being differentiation-stage specific. However, factor and remained at relatively invariant levels through the since the nonadherent monocytes had a 4-fold greater frac- remainder of the cell cycle (Fig. 3), as reported (38). tion of proliferating cells when compared to the adherent c-fgr mRNA was also induced when the adherent cells macrophages, these differences in gene expression could were stimulated to proliferate with CSF-1, but the time instead be related to the extent of proliferation at each course of induction was different from that of c-fos and differentiation stage. Day-6 culture medium is largely deplet- c-myc. As shown in Fig. 3, the 2.6-kb c-fgr transcript [and the ed ofCSF-1, since it has been used by the differentiating cells larger 3.5-kb and 4.7-kb transcripts previously described (25). In addition, nonadherent cells require only one-fifth of (14)], began to be detected 4 hr after growth-factor addition the CSF-1 concentration required by differentiated adherent (representing a 5-fold induction from baseline levels) in the G1 cells to proliferate (36). Thus, the differences in proliferation phase of the cell cycle and reached maximal levels at 8 hr in the two monocytic populations may have been in part due (representing a 20-fold induction). These maximal levels of to reduced levels of CSF-1 in the day-6 culture medium. c-fgr mRNA persisted to 16 hr and began to decline to CSF-1 is essential for the induction of proliferation in baseline levels by 20 hr, as the monocytic cells entered S differentiated monocytic cells and, in the absence of CSF-1, phase of the cell cycle (Fig. 3A). Under identical experimen- adherent cells enter a quiescent state (Go/G1), even in the tal conditions, there was no induction ofc-src or c-yes mRNA presence of serum (37). To determine whether c-fos and c-fgr transcripts by CSF-1 (data not shown). In contrast to c-fos, expression could be reinduced when the more highly differ- c-myc, and c-fgr, expression of c-fms and gpd (34) were entiated adherent monocytic cells were stimulated to prolif- detected at relatively invariant levels during the stimulation erate, these cells were synchronized in the Go/G1 phase ofthe of proliferation and cell-cycle progression (Fig. 3). cell cycle by removing the culture medium and incubating the Expression ofc-src and c-fgrin Myeloid Leukemia Cells. The cells for 24 hr in fresh medium supplemented only with fetal most immature cell that we have isolated from day-6 bone

% MAX. ABSBANOE * LABELLED NUCLEI A U)CC:v B Lo (N 0 b - T O e c-fos .

c- myc

c- fgr c-fms - * w

gpd _ as - o 4 8 12 16 20 24 28 32 38 40 14 HOURS 1 2 3 4 5 6 7 8 9 101112 13 HOURS

FIG. 3. CSF-1 stimulation of proliferation in monocytic cells induces c-fos, c-myc, and c-fgr gene expression. Day-6 adherent cells were rendered quiescent and synchronized in the Go/G, phase of the cell cycle by incubating them for 24 hr with fresh a-MEM containing only 10% fetal bovine serum. At 24 hr (time 0), the cultures were replated with fresh a-MEM containing 10% fetal bovine serum and stage IV-purified CSF-1 (2000 units/ml). Alternatively, the readdition offresh medium containing 10% fetal bovine serum and 10% L929 cell-conditioned medium containing crude CSF-1 gave identical results (data not shown). The entry of the monocytic cells into S phase was monitored by [3H]thymidine uptake and autoradiography as described (35). (A) Monocytic cells first began to enter S phase at -16 hr, with the majority of cells entering S phase by 22-24 hr. (B) Total RNA was also isolated from these cells after CSF-1 stimulation. Ten micrograms oftotal RNA from day-6 adherent cells (lane 1), from adherent cells rendered quiescent by removal of CSF-1 for 24 hr (lane 2, time 0), and from adherent cells stimulated to proliferate with CSF-1 at various time points from 0 to 36 hr (lanes 3-13) was analyzed by RNA blot hybridization. (C) Changes in c-fos (m), c-myc (+), and c-fgr (*) mRNA levels were determined by soft-laser scanning densitometry and are expressed as a percent of the maximal level obtained with each probe plotted against time. Downloaded by guest on September 23, 2021 Cell Biology: Willman et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4483 marrow cultures is the nonadherent cell that is already myelomonocytic phenotype. In contrast, the highly related committed to differentiation in the monocytic lineage. To gene c-src was expressed in both undifferentiated and differ- examine expression ofsrc- related sequences at earlier stages entiated myelomonocytic leukemia cells, while c-yes tran- of normal hematopoietic differentiation, we examined gene scripts were not detected in either normal or leukemic expression in acute myeloid leukemia cells. Since the cell- myelomonocytic cells at the level of sensitivity used in our surface antigenic phenotype of human leukemic cells has experiments. c-src, c-fgr, and c-yes are members of a larger been shown to be similar to the phenotype of cohort normal family of highly related cellular tyrosine kinases, which cells arrested at particular differentiation stages (39-41) and appear to have arisen by duplication from an ancestral src since leukemic cells appear to be a partial or complete gene. Other members of this family include c-syn, c-lyn, and stabilization of phenotypes that are transiently expressed in lck. While all of these genes encode proteins with highly equivalent normal cells (42), uncultured human leukemia homologous carboxyl-terminal kinase domains, the apparent cells represent a useful system in which to correlate patterns diversity in their amino-terminal domains suggests that each of gene expression with myeloid differentiation stages. We kinase may be independently regulated and may have unique have examined the expression of c-src, c-fgr, and c-yes in a substrate specificities. While c-src (11, 30), c-yes (16), and series of 45 clinical cases of acute myeloid leukemia (to be c-syn (16) are expressed in a wide variety of tissue types, published in detail elsewhere), which ranged in phenotype c-lyn (17), Ick (18), and c-fgr appear to display more lineage- from relatively undifferentiated myeloid leukemic blasts restricted patterns of expression; ick expression is largely [expressing HLA-Dr, the MylO human progenitor cell sur- restricted to T lymphocytes (18), while c-fgr is selectively face antigen (41), and the My7 myeloid cell-surface antigen expressed in differentiated cells of the myeloid lineage. The (39)] to blasts with a more differentiated myelomonocytic or low levels of c-fgr expression in undifferentiated myeloid monocytic phenotype [expressing HLA-Dr, My7, and the leukemia cells compared to differentiated cells suggests that My4 monocytic marker (39)]. Both leukemic cells with an c-fgr may not be expressed until later stages of myeloid undifferentiated myeloid progenitor cell phenotype (a repre- development. Despite the fact that Epstein-Barr virus- sentative case is shown in fig. 4A, lane 1) and with a more transformed lymphoid cells have been reported to contain differentiated myelomonocytic phenotype (Fig. 4B, lane 1) high levels of c-fgr mRNA (43), we and others (43) have been contained readily detectable levels ofthe 5.0-kb human c-src unable to detect c-fgr mRNA in human B- or T-cell-derived mRNA transcript. Differentiated leukemic cells contained lymphoid leukemias or in normal phytohemagglutinin-stim- 2-4 times lower levels of c-src mRNA than that observed in ulated peripheral blood T lymphocytes (unpublished obser- the undifferentiated cells (Fig. 4). In contrast, high levels of vations). the 3.5- and 2.6-kb c-fgr mRNA transcripts were detected The precise cellular functions of this family of tyrosine only in leukemic cells with a more differentiated myelomono- kinases remain to be elucidated. The c-src-encoded protein cytic phenotype (Fig. 4B, lane 2). Leukemic cells at both has been reported to be involved in the transduction of stages of differentiation contained similar levels of the con- proliferation signals both in transformed cells (4, 5) and in stitutively expressed gene gpd (Fig. 4 A and B, lanes 3). The normal fibroblasts (6). In contrast, the high levels ofpp6Oc-src differences in c-fgr and c-src expression in the leukemic cells protein and tyrosine kinase activity observed in nonprolifer- cannot be solely related to differences in proliferation at each ating cells and tissues, such as the nervous system (7-9), differentiation stage, since these two representative cases platelets (10), and myeloid lineage cells (44), imply that c-src had similar fractions of cells in S phase. The undifferentiated may have an important function in fully differentiated cells. leukemic cells shown in Fig. 4A had 70% ofcells in the Go/G1 However, the proliferation-related and differentiation-relat- phase of the cell cycle, 20.4% of cells in S phase, and 9.6% ed roles envisioned for these kinases may not be mutually of cells in G2/M, while the more differentiated myelo- exclusive. It is conceivable that these proteins may be monocytic blasts (Fig. 4B) had 79% of cells in Go/Gj, 13,9% components of a protein phosphorylation cascade that func- of cells in S phase, and 7.1% of cells in G2/M [determined by tions to transduce both proliferative as well as differentiation- flow cytometric techniques (26); data not shown]. related signals. Hematopoietic cells are unique in that certain differen- DISCUSSION tiated end-stage cells, such as monocytes and lymphocytes, retain their proliferative capacity in response to lineage- In our studies of the expression of src-related protoonco- specific growth factors. The induction of c-fgr mRNA expres- genes in myeloid cells, we have determined that c-fgr is sion in differentiated monocytes by CSF-1 is to our knowl- expressed at highest levels in differentiated myeloid cells, edge one of the first descriptions of the modulation of a both in normal bone marrow-derived cells committed to the src-related mRNA by a physiological growth factor. c-fgr monocytic lineage and in leukemic cells with a differentiated mRNA levels were induced 20-fold 8 hr after the addition of CSF-1 to quiescent normal monocytic cells and rapidly A B declined as the cells entered S phase (Fig. 3). Commitment to 1 2 3 1 23 the mitotic cell cycle is accomplished by the activation of

5.0kb w 5.0kb target proteins by phosphorylation and a cascade of phos- phorylation events has been associated with the G1 to S phase 2 6kb 26kb * transition (45). Similarly, commitment to the mitotic cell cycle in yeast is regulated by protein kinases homologous to the src family of tyrosine kinases (46). The induction of c-fgr FIG. 4. c-src and c-fgr expression in myeloid leukemia cells. (A) transcripts by CSF-1 leads us to speculate that c-fgr may also Ten micrograms of total RNA from uncultured human myeloid function in an analogous manner in the regulation of cell- leukemia cells with the progenitor cell phenotype (HLA-Dr', cycle progression in normal monocytic cells. Alternatively, My1O+, My7', My4-) were hybridized with probes specific for c-src the decrease in c-fgr expression observed as the cells enter S (lane 1), c-fgr (lane 2), and gpd (lane 3). (B) RNA from leukemia cells phase may imply that the levels and activity of this protein with a differentiated monocytic phenotype (HLA-Dr', MylO-, My7', My4+) was hybridized with probes for c-src (lane 1), c-fgr kinase must decrease as cells progress toward the S phase of (lane 2), and gpd (lane 3). The relative abundance of these two highly the cell cycle. related transcripts is reversed with monocytic differentiation in The signals triggered by the binding of myeloid CSFs such leukemic cells, while the constitutive gene gpdis expressed at similar as CSF-1 are complex. Although CSFs are an absolute levels. requirement for every round of proliferation in myeloid cells, Downloaded by guest on September 23, 2021 4484 Cell Biology: Willman et al. Proc. Natl. Acad. Sci. USA 84 (1987)

they also function to promote survival and to stimulate 17. Yamanashi, Y., Fukushige, S., Semba, K., Miyajima, N., functional activities in end-stage cells (23). Thus, our obser- Matsubara, K., Yamamoto, T. & Toyoshima, K. (1987) Mol. vations do not preclude the c-fgr-encoded protein from Cell. Biol. 7, 237-243. having a role in transducing differentiation-inducing signals in 18. Marth, J. D., Peet, R., Krebs, E. G. & Perlmutter, R. M. (1985) Cell 43, 393-404. monocytic cells. The system of developing normal bone 19. Boettinger, D., Anderson, S. & Dexter, T. M. (1984) Cell 36, marrow-derived monocytic cells that we have described in 763-773. this report will be useful for further characterization of the 20. Cook, W. D., Metcalf, D., Nicola, N. A., Burgess, A. W. & c-fgr gene, in determining the intracellular location and Walker, F. (1985) Cell 41, 677-683. function of its gene product, and in studies of the regulation 21. Pierce, J. H., Di Fore, P. P., Aaronson, S. A., Potter, M., of its expression and activation. Pumphrey, J., Scott, A. & IhIe, J. N. (1985) Cell 41, 685-693. 22. Carmier, J. F. & Samarut, J. (1986) Cell 44, 159-165. We would like to acknowledge the National Flow Cytometry 23. Metcalf, D. (1985) Science 229, 16-22. Resource at Los Alamos National Laboratories and particularly Dr. 24. Walker, E. B., Akporiaye, E., Warner, N. L. & Stewart, Harry Crissman and Dr. John Steinkamp for assistance in flow C. C. (1985) J. Leuk. Biol. 37, 121-136. cytometric studies. We would also like to thank Joan Brugge and Ray 25. Tushinski, R. J., Oliver, I. J., Guilbert, C. J., Tynan, P. W., Erikson for helpful discussions; the laboratories of J. M. Bishop, D. Warner, J. R. & Stanley, E. R. (1982) Cell 28, 71-81. Fugita, K. Robbins, H. Hanafusa, T. Papas, and A. Dugaiczyk for 26. Crissman, H. A., Darzynkiewicz, Z., Tobey, R. A. & Stein- the gifts ofDNA probes; Hsiu-san Lin for the generous gift ofpurified kamp, J. A. (1985) J. 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