Nucleolar Arf sequesters Mdm2 and activates p53.

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.

The product of the c-fms proto-oncogene: a glycoprotein with associated tyrosine kinase activity.

The c-fms proto-oncogene is a member of a gene family that has been implicated in tumorigenesis. Glycoproteins encoded by c-fms were identified in cat spleen cells by means of an immune-complex kinase assay performed with monoclonal antibodies to v-fms-coded epitopes. The major form of the normal cellular glycoprotein has an apparent molecular weight of 170,000 and, like the product of the viral oncogene, serves as a substrate for an associated tyrosine-specific protein kinase activity in vitro. The results suggest that the transforming glycoprotein specified by v-fms is a truncated form of a c-fms-coded growth factor receptor.

Ink4c is dispensable for tumor suppression in Myc-induced B-cell lymphomagenesis.

p18(Ink4c) functions as a dedicated inhibitor of cyclin-D-dependent kinases. Loss of Ink4c predisposes mice to tumor development and, in a dose-dependent manner, complements the tumor-promoting effects of various oncogenes. We have now addressed whether Ink4c loss impacts B-cell tumor development in the Emu-Myc transgenic mouse, a model of human Burkitt lymphoma. Loss of one or both alleles did not influence the onset of lymphoma in Emu-Myc transgenics, and did not appreciably affect Myc's proliferative or apoptotic responses in precancerous B cells. Nevertheless, Ink4c loss modulated the effects of Myc-induced transformation by decreasing the frequency of Arf loss, an ordinarily common event in Emu-Myc-induced lymphomas.

Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells.

Expression of the human c-fms proto-oncogene product (colony-stimulating factor-1 receptor) on peripheral blood mononuclear cells and choriocarcinoma cell lines.

The c-fms gene product is related, and possibly identical, to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. Using antisera to a recombinant v-fms--coded polypeptide, glycoproteins encoded by the human c-fms locus were detected in mononuclear cells from normal peripheral blood and in promyelocytic HL-60 cells 24 h after induction of monocytic differentiation with phorbol ester. The 150-kD human c-fms--coded glycoprotein was expressed at the cell surface, was active as a tyrosine-specific protein kinase in vitro, and shared primary structural features with the product of the feline retroviral v-fms oncogene. A biochemically indistinguishable glycoprotein was detected in human choriocarcinoma cell lines. Like peripheral blood mononuclear cells and phorbol ester-treated HL-60 cells, the choriocarcinoma cells expressed high affinity binding sites for human CSF-1. In addition to serving as a lineage specific growth factor in hematopoiesis, CSF-1 may play a role in normal trophoblast development.

Transfer and expression of the gene encoding a human myeloid membrane antigen (gp150).

DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.

Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.

Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms.

The turnover of the colony-stimulating factor 1 receptor (CSF-1R), the c-fms proto-oncogene product, is accelerated by ligand binding or by activators of protein kinase C (PKC), such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The mechanisms of ligand- and TPA-induced downmodulation were shown to differ by the following criteria. First, in cells in which PKC was downmodulated, CSF-1R reexpressed at the cell surface remained sensitive to ligand but was refractory to TPA-induced degradation. Second, a kinase-defective receptor containing a methionine-for-lysine substitution at amino acid 616 at its ATP-binding site failed to undergo ligand-induced downmodulation but remained responsive to TPA. Following CSF-1 stimulation, no intermediates of receptor degradation could be immunoprecipitated with polyvalent antisera to CSF-1R. In contrast, TPA induced specific proteolytic cleavage of the receptor near its transmembrane segment, resulting in the release of the extracellular ligand-binding domain from the cell and the generation of an intracellular fragment containing the kinase domain. Two-dimensional phosphopeptide mapping demonstrated no new sites of phosphorylation in response to TPA in either the residual intact receptor or the intracellular proteolytic fragment. Therefore, PKC appears not to trigger downmodulation by directly phosphorylating the receptor but, rather, activates a protease which recognizes CSF-1R as a substrate.

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.

The oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3 homeodomain recognition helix but not the Pax3 paired-box DNA binding domain.

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third alpha-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis.

The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in E mu-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in E mu-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type E mu-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions in ARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in E mu-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective of Bax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas from Bax-null E mu-myc transgenics lacked p53 alterations, whereas 27% of the tumors in Bax(+/-) E mu-myc transgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection of p53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily the case for ARF and Mdm2.

Apoptosis triggered by Myc-induced suppression of Bcl-X(L) or Bcl-2 is bypassed during lymphomagenesis.

Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-X(L) or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-X(L) RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-X(L) and Bcl-2 expression is inhibited by Myc in precancerous B cells from Emu-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-X(L) by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-X(L) levels are markedly elevated in over one-half of all lymphomas arising in Emicro-myc transgenic mice. Bcl-2 and/or Bcl-X(L) overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-X(L) or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.

INK4d-deficient mice are fertile despite testicular atrophy.

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.

Ligand-induced phosphorylation of the colony-stimulating factor 1 receptor can occur through an intermolecular reaction that triggers receptor down modulation.

Ligand-induced tyrosine phosphorylation of the human colony-stimulating factor 1 receptor (CSF-1R) could involve either an intra- or intermolecular mechanism. We therefore examined the ability of a CSF-1R carboxy-terminal truncation mutant to phosphorylate a kinase-defective receptor, CSF-1R[met 616], that contains a methionine-for-lysine substitution at its ATP-binding site. By using an antipeptide serum that specifically reacts with epitopes deleted from the enzymatically competent truncation mutant, cross-phosphorylation of CSF-1R[met 616] on tyrosine was demonstrated, both in immune-complex kinase reactions and in intact cells stimulated with CSF-1. Both in vitro and in vivo, CSF-1R[met 616] was phosphorylated on tryptic peptides identical to those derived from wild-type CSF-1R, suggesting that receptor phosphorylation on tyrosine can proceed via an intermolecular interaction between receptor monomers. When expressed alone, CSF-1R[met 616] did not undergo ligand-induced down modulation, but its phosphorylation in cells coexpressing the kinase-active truncation mutant accelerated its degradation.

Transduction of human colony-stimulating factor-1 (CSF-1) receptor into interleukin-3-dependent mouse myeloid cells induces both CSF-1-dependent and factor-independent growth.

A retroviral vector encoding the receptor for human colony-stimulating factor-1 (CSF-1) was introduced into murine myeloid FDC-P1 cells which require interleukin-3 (IL-3) for their proliferation and survival in culture. Cells expressing the CSF-1 receptor (CSF-1R), selected by fluorescence-activated cell sorting in the continued presence of murine IL-3, formed colonies in semisolid medium and were able to proliferate continuously in liquid cultures containing human recombinant CSF-1. Thus, although they do not synthesize endogenous murine CSF-1R, FDC-P1 cells express the downstream components of the CSF-1 mitogenic pathway necessary for its signal-response coupling. After receptor transduction, slowly proliferating factor-independent variants that produced neither CSF-1 nor growth factors able to support the proliferation of parental FDC-P1 cells also arose. When the human CSF-1R was expressed in FDC-P1 cells under the control of an inducible metallothionein promoter, the frequencies of both CSF-1-responsive and factor-independent variants increased after heavy-metal treatment. In addition, a monoclonal antibody to human CSF-1R arrested colony formation by both the CSF-1-dependent and factor-independent cells but did not affect their growth in response to IL-3. Therefore, the induction of both the CSF-1-dependent and factor-independent phenotypes depended on expression of the transduced human CSF-1R.

Cell surface expression of v-fms-coded glycoproteins is required for transformation.

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.

Inhibition of cell proliferation by the Mad1 transcriptional repressor.

Mad1 is a basic helix-loop-helix-leucine zipper protein that is induced upon differentiation of a number of distinct cell types. Mad1 dimerizes with Max and recognizes the same DNA sequences as do Myc:Max dimers. However, Mad1 and Myc appear to have opposing functions. Myc:Max heterodimers activate transcription while Mad:Max heterodimers repress transcription from the same promoter. In addition Mad1 has been shown to block the oncogenic activity of Myc. Here we show that ectopic expression of Mad1 inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1) receptor. The ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F (containing a Y-to-F mutation at position 809) is also inhibited by Mad1. Cell cycle analysis of proliferating 3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 which inhibit its activity as a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1 to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor.

A potential role for Elf-1 in terminal transferase gene regulation.

The terminal deoxynucleotidyltransferase (TdT) gene represents an attractive model for the analysis of gene regulation during an early phase of lymphocyte development. In previous studies, we identified a DNA element, termed D', which is essential for TdT promoter activity in immature lymphocytes, and two classes of D'-binding factors, Ikaros proteins and Ets proteins. Here, we report a detailed mutant analysis of the D' element which suggests that an Ets protein, rather than an Ikaros protein, activates TdT transcription. Since multiple Ets proteins are expressed in developing lymphocytes and are capable of binding to the D' element, DNA affinity chromatography was used to determine if one of the Ets proteins might bind to the D' element with a uniquely high affinity, thereby implicating that protein as a potential TdT activator. Indeed, one binding activity was greatly enriched in the high-salt eluates from a D' affinity column. Peptide microsequencing revealed that the enriched protein was Elf-1. Immunoblot analyses confirmed that in nuclear extracts, Elf-1 has a significantly higher affinity for the D' sequence than does another Ets protein, Ets-1. Transactivation and expression studies support the hypothesis that Elf-1 activates TdT transcription in immature T and B cells. Finally, a D' mutation which selectively reduces Elf-1 binding, but not the binding of other Ets proteins, was found to greatly reduce TdT promoter activity. Although Elf-1 previously had been implicated in the inducible activation of genes in mature T and B cells, our results suggest that it also plays an important role in regulating genes during an early phase of lymphocyte development.

Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression.

The activity of p21ras is required for the proliferative response to colony-stimulating factor 1 (CSF-1), and signals transduced by both the CSF-1 receptor (CSF-1R) and p21ras stimulate transcription from promoter elements containing overlapping binding sites for Fos/Jun- and Ets-related proteins. A sequence encoding the DNA-binding domain and nuclear localization signal of human c-ets-2, which lacked portions of the c-ets-2 gene product necessary for trans activation, was fused to the bacterial lacZ gene and expressed from an actin promoter in NIH 3T3 cells expressing either the v-ras oncogene or human CSF-1R. Nuclear expression of the Ets-LacZ protein, confirmed by histochemical staining of beta-galactosidase, inhibited the activity of ras-responsive enhancer elements and suppressed morphologic transformation by v-ras as well as CSF-1R-dependent colony formation in semisolid medium. When CSF-1R-bearing cells expressing the Ets-LacZ protein were stimulated by CSF-1, induction of c-ets-2, c-jun, and c-fos ensued, but the c-myc response was impaired. Enforced expression of the c-myc gene overrode the suppressive effect of ets-lacZ and restored the ability of these cells to form colonies in response to CSF-1. NIH 3T3 cells engineered to express a CSF-1R (Phe-809) mutant similarly cannot form CSF-1-dependent colonies in semisolid medium and exhibit an impaired c-myc response, but expression of an exogenous myc gene resensitizes these cells to CSF-1 [M. F. Roussel, J. L. Cleveland, S. A. Shurtleff, and C. J. Sherr, Nature (London) 353:361-363, 1991]. The ability of these cells to respond to CSF-1 was also rescued by enforced expression of an endogenous c-ets-2 gene. The ets family of transcription factors therefore plays a central role in integrating both CSF-1R and ras-induced mitogenic signals and in modulating the myc response to CSF-1 stimulation.