Genetic dissection of the control of normal differentiation in myeloid leukemic cells

Normal myeloid precursors and MGI(+)D(+) myeloid leukemic cells can be induced to differentiate to mature cells by the normal protein inducer MGI. The sequence of differentiation is the induction of C3 and Fc rosettes, C3 and Fc immune phagocytosis (IP), synthesis and secretion of lysozyme, and formation of mature macrophages and granulocytes. Mutant clones of myeloid leukemic cells have been isolated with differences in the time of induction of C3 and Fc rosettes and C3 and Fc IP, in which lysozyme was induced without going through the stage of Fc or C3 IP, and with differences in inducibility by MGI to mature macrophages or granulocytes. Only one out of five MGI(-)D(-) clones gave rise to MGI(+)D(+) mutants. The ability to obtain mutants from this clone was associated with its special chromosome constitution, and these mutants showed a change in their ability for cap formation by concanavalin A. The steroid inducer dexamethasone can induce in MGI(+)D(+) clones differentiation to macrophages but not to granulocytes. Differentiation by steroid inducer in different clones occurred either with or without induction of Fc rosettes and Fc IP, and induction of C3 rosettes was not always associated with induction of C3 IP. The use of mutants that differ in their competence to be induced by MGI or steroid inducer has shown that there are separate controls for the induction of C3 and Fc rosettes, C3 and Fc IP, lysozyme, macrophages, and granulocytes.     

In vivo induction of normal differentiation in myeloid leukemia cells

MGI(+)D(+), MGI(+)D(-), and MGI(-)D(-) mouse myeloid leukemic cells, which genetically differ in their competence to be induced to undergo normal cell differentiation in vitro by the normal macrophage- and granulocyte-inducing protein MGI, were analyzed for their ability to undergo cell differentiation in diffusion chambers in vivo. As after induction by MGI in vitro, MGI(+)D(+) clones were induced for Fc and C3 rosettes, lysozyme, and mature macrophages and granulocytes in normal syngeneic or allogeneic mice. MGI(+)D(-) clones were also induced in these mice for all these properties, although in vitro they were not induced by MGI for mature cells. The MGI(-)D(-) clones were induced in vivo for C3 and Fc rosettes, lysozyme, and intermediate stages but not for mature cells, whereas none of these properties were induced in these clones by MGI in vitro. Thus, certain types of myeloid leukemic cells differentiate better in vivo, possibly due to the presence of higher effective concentrations of MGI and/or other inducing factors, and MGI(+)D(+) and MGI(+)D(-) cells can completely differentiate in vivo to mature cells. In vivo differentiation was inhibited in mice treated with cyclophosphamide. It was also inhibited in various strains of nude mice, except for one MGI(+)D(+) clone, where it was inhibited in C57BL/6 but not in ICR nude mice. This MGI(+)D(+) clone was also the only clone that was induced to differentiate normally in vitro by a 23,000 molecular weight form of purified MGI. The results suggest that different clones respond to different molecular forms of MGI, which may be present in different proportions in some animals, that in vivo differentiation by MGI possibly with other factors may be regulated by cells involved in the immune response, and that this differentiation can be genetically controlled. Differentiation in vivo was enhanced by injection of conditioned medium containing MGI and by inoculation of MGI-producing cells, including normal granulocytes. This indicates that the induction of normal differentiation of myeloid leukemic cells in vivo can be enhanced by these treatments.     

Differential desensitization of functional adrenergic receptors in normal and malignant myeloid cells: Relationship to receptor-mediated hormone cytotoxicity

Malignant myeloid leukemic cells and normal macrophages and granulocytes have functional beta-adrenergic receptors, which have been quantitated by radioreceptor binding with the beta-adrenergic antagonist [(3)H]dihydroalprenolol and by induction of cyclic AMP by adrenergic hormones. Both the normal and leukemic cells have beta(2)-adrenergic receptors, and the [(3)H]dihydroalprenolol binding was saturable, reversible, and stereospecific. The leukemic cells consisted of clones that could be induced to differentiate (MGI(+)D(+)) and clones that could not be induced to differentiate to mature macrophages and granulocytes by the protein inducer MGI. The different types of leukemic clones all had 1100-2300 receptor sites per cell, whereas normal macrophages had 7000 receptors per cell. The differentiation of MGI(+)D(+) leukemic cells was associated with an increase in receptors to a number similar to that found with normal macrophages. MGI(+)D(+) leukemic cells and normal macrophages were able to densensitize to the beta-adrenergic agonist (-)isoproterenol, shown by termination of cyclic AMP induction within 10-15 min and the lack of a second induction. The leukemic cells that could not be induced to differentiate lacked this capacity for desensitization, possibly due to an alteration in the uncoupling system between the receptor and adenylate cyclase. The lack of desensitization in these leukemic cells was associated with a higher sensitivity to the receptor-mediated cytotoxic effects of adrenergic hormones. It is suggested that cells, like some leukemic cells, that are unable to desensitize to adrenergic and possibly other hormones may be appropriate targets for differential destruction by hormones under conditions that do not affect normally desensitizing cells.     

Different Blocks in the Differentiation of Myeloid Leukemic Cells

Some clones of mouse myeloid leukemic cells (D(+)) can be induced to undergo cell differentiation to mature macrophages and granulocytes, and other clones (D(-)) could not be induced to differentiate to mature cells. Normal mature macrophages and granulocytes have surface receptors that form rosettes with erythrocytes coated with specific immunoglobulin or immunoglobulin-complement. The D(+) clones were induced to form receptors by prednisolone, cytosine-arabinoside, 5-iododeoxyuridine, actinomycin D, or serum from mice injected with endotoxin. All these compounds thus induced a common change in the cell surface membrane. The induction of receptors required protein synthesis, and receptors were formed before the appearance of mature cells. There were two types of D(-) clones. One type was induced by these compounds to form receptors, although with a lower inducibility than D(+) clones; in the other type there was no induction of receptors. The results indicate that there are different blocks in the differentiation of myeloid leukemic cells. Some leukemic cells (IR(+)D(+)) can be induced to form receptors and to differentiate to mature cells; others (IR(+)D(-)) can form receptors but not mature cells; and a third type (IR(-)D(-)) could not be induced to form receptors or mature cells.     

Control of Normal Differentiation of Myeloid Leukemic Cells to Macrophages and Granulocytes

Cells from a myeloid leukemic line in culture can be induced by the differentiation-inducing protein MGI to form colonies with normal differentiation to mature macrophages and granulocytes. This line consisted of clones that can be induced to undergo normal cell differentiation (D(+) clones) and clones (D(-) clones) that were not inducible. D(+) clones were able to undergo differentiation to both macrophages and granulocytes. Normal differentiation was induced even in clones that were no longer diploid. D(+) clones can segregate some D(-) progeny, and D(-) clones can segregate some D(+) progeny. This, therefore, provides a system for studies on the genetic and chemical control of cell differentiation in leukemic cells.     

Increase of normal myeloblast viability and multiplication without blocking differentiation by type C RNA virus from myeloid leukemic cells.

Clones of mouse myeloid leukemic cells that differ in their competence to be induced for normal cell differentiation by the protein inducer MGI produce type C virus. These viruses have been studied for their effect on the viability, multiplication, and differentiation of normal bone marrow cells either with or without the addition of MGI. Virus from leukemic clones that can differentiate normally to mature macrophages and granulocytes (MGI+D+ clones) induced some multiplication of myeloblasts in the bone marrow, but the cells did not differentiate without adding MGI. In the presence of MGI, this virus then induced an increased number of colonies whose cells differentiated to mature macrophages or granulocytes as in colonies of uninfected cells. Virus infection also resulted in a decrease in the amount of MGI and fetal calf serum that was required for colony formation. Virus from MGI+D+ clones, in the presence of MGI, was 500-fold more effective in increasing colony formation than virus from the differentiation-defective MGI-D- clones, although both types of virus replicated with equal efficiency in the normal bone marrow cells. No such increase was obtained after infection with the Friend leukemic virus complex or the Moloney murine leukemia virus. Infection with virus from a MGI+D+ clone that was differentiated by MGI mainly to macrophages induced a higher percentage of macrophage colonies than virus from MGI+D+ clones that were differentiated by MGI to granulocytes and macrophages. Studies with isolated myeloblast colony-forming cells from the bone marrow have indicated that these are the target cells for the virus. Infections of these isolated myeloblasts with virus from MGI+D+ clones induced some multiplication without differentiation in the absence of MGI, and increased the viability and multiplication of the myeloblasts without inhibiting their ability to differentiate in the presence of MGI. The results, therefore, indicate that virus from MGI+D+ cells can increase the viability and multiplication of normal myeloblasts in the bone marrow without blocking the ability of these cells to be induced to differentiate by MGI, and that this effect was directly related to the competence of the leukemic host cells to be induced for normal differentiation. It is suggested that the difference between the effect of virus from MGI+D+ and MGI-D- cells may be due to a difference in their integration sites in relation to the genes that control cell viability, multiplication, and differentiation.     

Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion

The control of cell multiplication and differentiation by tumor-promoting phorbol esters including 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied with different clones of mouse myeloid leukemic cells, a line of human myeloid leukemic cells, and normal mouse bone marrow myeloblasts. TPA induced normal cell differentiation in one of the mouse leukemic clones and this was mediated by induction of the protein inducer of differentiation to macrophages or granulocytes (MGI) in the cells that then differentiated. Other mouse clones were not induced to differentiate by TPA. In one of these clones, TPA induced cell susceptibility to externally added MGI. This effect was not due to a general induction of susceptibility to all compounds because TPA did not induce susceptibility to lypopolysaccharide or dexamethasone in this clone. In the human leukemic cell line, TPA also induced differentiation with the induction of MGI activity and enhanced susceptibility to added MGI. It is suggested that the clonal differences in induction of MGI activity and increased susceptibility to MGI may be associated with differences in receptors for TPA and the ability of TPA to modify receptors for MGI. Studies with normal bone marrow cells have indicated that TPA stimulated MGI activity and also increased susceptibility of normal myeloblasts to induction of multiplication by MGI. The ability of different phorbol esters to produce these effects on normal myeloblasts and myeloid leukemic cells paralleled their ability to act as tumor promoters. The results indicate that a tumor promoter such as TPA can induce the production of and increase cell susceptibility to a normal regulator of cell multiplication and differentiation. TPA has pleiotropic effects. It is suggested that, by these mechanisms, TPA may thus act as a tumor promoter by increasing cell multiplication in initiated cells, induce differentiation in some cells, or inhibit differentiation in other cells, depending on which molecules are being regulated in the TPA-treated cells.     

Chromosome mapping of the genes that control differentiation and malignancy in myeloid leukemic cells.

The chromosome banding pattern has been analyzed in clones of mouse myeloid leukemic cells that differ in their ability to be induced to differentiate by the protein inducer MGI (macrophage and granulocyte inducer). None of the clones had a completely normal diploid banding pattern. The clones studied were either MGI+ (that can be induced to form Fc and C3 rosettes), a stage in the differentiation of myeloid cells, or MGI- (that cannot be induced to form these rosettes). All six cultured clones of MGI- cells from myeloid leukemias independently produced in six separate animals showed a loss of a piece of one chromosome 2 and this abnormal chromosome was maintained in leukemias derived from the cultured cells. This loss was not found in MGI+ clones or lymphoid leukemias. Five MGI+ mutants, derived from an MGI- clone with a loss of a piece of one chromosome 2, one normal chromosome 12, and two translocated chromosomes 12, maintained the abnormal chromosome 2 but lost either the one normal or one of these translocated chromosome 12. These results indicate that chromosomes 2 and 12 carry genes that control the differentiation of myeloid leukemic cells and that inducibility by MGI is controlled by the balance between these genes. We suggest that these chromosomes also carry genes that control the malignancy of these cells.     

Selective regulation of the activity of different hematopoietic regulatory proteins by transforming growth factor beta 1 in normal and leukemic myeloid cells

The viability of normal bone marrow myeloid precursor cells induced by interleukin-6 (IL-6) or IL-1 alpha and the ability of IL-6 and IL-1 alpha to induce the formation of colonies of granulocytes, macrophages, or megakaryocytes in densely seeded bone marrow cultures was suppressed by transforming growth factor-beta 1 (TGF-beta 1). Induction of normal bone marrow colony formation by IL-3 was much less sensitive to TGF-beta 1, and there was little or no effect of TGF-beta 1 on colony formation induced by macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage CSF (GM-CSF). In different clones of myeloid leukemic cells, TGF-beta 1 suppressed differentiation induced with IL-6, IL-1 alpha, or lipopolysaccharide (LPS), but did not suppress differentiation induced with IL-3 or GM-CSF. The effect of TGF-beta 1 on differentiation of the leukemic cells can be dissociated from its effect on cell growth. TGF-beta 1 suppressed the production of IL-6 in normal bone marrow cells cultured with IL-1 alpha and the production of IL-6 and GM-CSF in leukemic cells cultured with IL-1 alpha or LPS. The suppression of IL-6 production can explain the suppression by TGF-beta 1 of the effects of IL-1 alpha and LPS that are mediated by IL-6. TGF-beta 1 also suppressed differentiation in clones of myeloid leukemic cells induced with differentiation factor/leukemia inhibitory factor and tumor necrosis factor. In different leukemic clones TGF-beta 1 suppressed or enhanced induction of differentiation with dexamethasone. The results show that TGF-beta 1 can selectively control the activity of different molecular regulators of normal and leukemic hematopoiesis.     

Mobility of Carbohydrate-Containing Structures on the Surfaces Membrane and the Normal Differentiation of Myeloid Leukemic Cells to Macrophages and Granulocytes

Clones (D(+)) of a cultured line of myeloid leukemic cells can be induced to undergo normal differentiation to mature macrophages and granulocytes. There are also clones derived from the same cell line (D(-)) that could not be induced to differentiate. The carbohydrate-binding protein concanavalin A was used as a probe to study the mobility of carbohydrate-containing sites on the surface membrane of these cells. Changes in the distribution of concanavalin A binding sites on the surface membrane can be induced by concanavalin A. With the appropriate site mobility, this induction of a new distribution resulted in a concentration of concanavalin A-membrane site complexes on one pole of the cell to form a cap. D(+) and D(-) clones showed 50 and 5% of cells with caps, respectively, although both types of cells bound a similar number of concanavalin A molecules. Treatment of cells with trypsin increased cap formation from 5 to 40% in D(-) cells, but did not change the percentage of cells with caps in D(+) cells. The results show a difference in the mobility of concanavalin A binding sites in these two types of cells and suggest a difference in the fluid state of these carbohydrate-containing structures on the surface membrane. It is suggested that a gain of the ability of myeloid leukemic cells to undergo normal differentiation is associated with an increase in the fluidity of structures on the surface membrane where the concanavalin A sites are located. Differences in fluidity of specific membrane sites may also explain differences in the response of cells to other differentiation-inducing stimuli.     

Evidence that G-CSF is a fibroblast growth factor that induces granulocytes to increase phagocytosis and to present a mature morphology, and that macrophages secrete 45-kd molecules with these activities as well as with G-CSF-like activity.

Evidence is provided that conditioned medium from a macrophage-like cell line contains molecules of approximately 45 kd molecular weight with granulocyte colony-stimulating factor (G-CSF)-like activity as well as with the property of inducing granulocytes to phagocytose latex particles and to mature morphologically. This type of differentiation was found to be induced on either bone marrow or induced granulocytes, but not on resident or induced macrophages. On the other hand, resident but not induced macrophages are shown to induce these types of activities when challenged by bacterial lipopolysaccharides. Evidence that macrophages produce a factor that is mitogenic for fibroblasts is also provided. This activity was measured by the induction of increased proliferation by either low-density or saturated cultures of fibroblasts. Human recombinant G-CSF was employed and found also to possess these dual capabilities of inducing both the proliferation and differentiation of granulocytes as well as the proliferation of fibroblasts. Finally, a mechanism for the regulation of myeloid cell production and differentiation is described in which G-CSF produced by macrophages not only induces granulocytes to differentiate but induces fibroblasts to proliferate and secrete macrophage colony-stimulating factor (M-CSF), which in turn makes myeloid monocyte precursors proliferate and secrete more G-CSF.     

Inhibition of differentiation and affinity purification with a monoclonal antibody to a myeloid cell differentiation-inducing protein

The normal myeloid hematopoietic regulatory proteins include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL-3), or macrophage and granulocyte inducers, type 1 (MGI-1), and another type of protein (MGI-2) with no myeloid cell growth-inducing activity that induces differentiation of normal myeloid precursor cells and certain clones of myeloid leukemic cells. An IgG2a monoclonal antibody was prepared and it neutralized two forms of MGI-2 (MGI-2A and MGI-2B) produced by mouse Krebs ascites tumor cells. The monoclonal antibody was used for affinity purification of MGI-2. This antibody also neutralized MGI-2 produced by normal mouse macrophages, normal myeloblasts incubated with IL-3, and MGI-2 produced by the lungs and found in the serum of mice injected with lipopolysaccharide (LPS). The anti-MGI-2 antibody did not inhibit the activity of any one of the four myeloid growth-inducing proteins (CSF or IL-3 = MGI-1), IL-1, tumor necrosis factor, or lymphotoxin. This antibody also inhibited induction of differentiation of myeloid leukemic cells by LPS, which is mediated by the endogenous production of MGI-2, but did not inhibit induction of differentiation in these leukemic cells by dexamethasone or cytosine arabinoside, which is not mediated by MGI-2. Anti-MGI-2 antibody thus inhibited differentiation when MGI-2 was added externally to cells or when it was mediated by endogenously produced MGI-2.     

Intracellular Lysozyme in Mature Neutrophils and Blast Cells in Acute Leukemia

Intracellular lysozyme (muramidase) activity was measured in leukemic blastsand mature neutrophilic granulocytes from20 patients with acute myeloblastic andmyelomonocytic leukemia and in 11 patients with acute lymphoblastic leukemiaafter differential centrifugation of cellsin Ficoll and extraction of lysozyme withn-butanol. Considerable abnormalities incellular lysozyme activity were found bothin qualitative and quantitative terms. Incontrast to normal myeloblasts, leukemicblasts of the myeloid series containedlysozyme in a considerable number ofcases. Although no clear-cut distinctionwas seen, those patients with positiveblast lysozyme reactivity tended to havethe highest plasma lysozyme levels,whereas no good correlation was foundbetween morphologic differentiationalong myeloblastic or monocytoblasticlines of blasts and lysozyme reactivity.Calculations of the magnitude of lysozymeproduction in acute leukemias with highplasma lysozyme concentration was compatible with the hypothesis that in thesecases lysozyme must be secreted by intactblasts and that, consequently, plasmalysozyme activity reflects the total leukemic cell mass. In mature neutrophilicgranulocytes from patients with acutemyeloblastic and myelomonocytic leukemia in relapse, the mean lysozyme activity was significantly decreased, although a great deal of variation wasfound. In remission, neutrophil lysozymeactivity seemed to increase; among several possibilities this might be a reflectionof different clones being operative in relapse and remission. In acute lymphoblastic leukemia, lysozyme activity inneutrophils was constantly low in relapseand increased to normal following induction of remission, which may be themain explanation of the low plasmalysozyme activity found in this type ofacute leukemia. It is unexplained andpuzzling why intraneutrophil lysozymeactivity is low in a leukemic type wherethe myeloid cells are not believed to beprimarily leukemic; one possible reasonmight be an effect of cell-to-cell interaction with the leukemic cell population.

Submitted on November 30, 1973 Revised on February 19, 1974 Accepted on February 20, 1974     

Induction of dependence on hematopoietic proteins for viability and receptor upregulation in differentiating myeloid leukemic cells

There are different types of myeloid leukemic cells that can be induced to differentiate to mature granulocytes or macrophages by different hematopoietic regulatory proteins. One type of leukemic clone can be induced to differentiate by recombinant macrophage and granulocyte differentiation-inducing protein-type 2 (MGI-2), which we have shown is Interleukin-6 (IL-6), and another type of leukemic clone can be differentiated by recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3. There was no subpopulation of growth factor- responsive or differentiation-defective cells before induction of differentiation in either type of clone. In both clones, induction of differentiation-induced requirement for a hematopoietic protein for cell viability. Viability of the cells was maintained by IL-6, IL-3, or macrophage colony-stimulating factor (M-CSF) but not by GM-CSF in the cells differentiated by IL-6, and by GM-CSF or IL-3 but not by IL-6 or M-CSF in the cells differentiated by GM-CSF or IL-3. The viable cells with a differentiated phenotype continued to multiply. In undifferentiated leukemic cells with no or few surface receptors for some of these proteins, there was an upregulation of the number of receptors during differentiation for the proteins to which the cells responded. But there were also differentiating leukemic cells with an upregulation of GM-CSF receptors although GM-CSF could not maintain the viability of the differentiating cells. The results indicate that induction of hormone responsiveness and upregulation of the hormone receptors can both occur in differentiating leukemic cells, and that the regulation of these two events can be separated.     

Desensitization of enucleated cells to hormones and role of cytoskeleton in control of normal hormonal response.

Prostaglandin E1 and the beta-adrenergic hormone l-isoproterenol stimulated cyclic AMP formation in both nucleated and enucleated myeloid leukemic cells that could be induced to differentiate normally to mature cells by the macrophage- and granulocyte-inducing protein MGI (MGI+D+ cells). Enucleated as well as nucleated MGI+D+ cells also desensitized to these hormones, indicating that this desensitization is an extranuclear process. Nucleated or enucleated mutant myeloid leukemic cells that are not induced to differentiate (MGI-D- cells) were not desensitized to these hormones. The antitubulin alkaloids colchicine and vinblastine, but not the antimicrofilament compound cytochalasin B, increased the maximal hormone-induced formation of cyclic AMP in nucleated MGI+D+ cells but not in the MGI-D- cells. These alkaloids also inhibited the development of desensitization to l-isoproterenol and prostaglandin E1 in enucleated MGI+D+ cells. The results indicate that in MGI+D+ cells the cytoskeletal system puts constraints on the cells' ability to respond to these hormones and that these constraints are absent in the mutant MGI-D- cells. Because MGI+D+ but not MGI-D- cells can be induced to differentiate by the macrophage- and granulocyte-inducing protein, cytoskeletal constraints, which are also found in normal myeloid cells, may be necessary for cell competence to differentiate. The results support the suggestion that membrane cytoskeletal constraints generate may control the normal response and desensitization to membrane-mediated cell inducers.     

In vivo control of differentiation of myeloid leukemic cells by cyclosporine A and recombinant interleukin-1 alpha

There are different types of hematopoietic regulatory proteins that regulate the multiplication and differentiation of normal myeloid cells. These different types include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL- 3), or macrophage and granulocyte inducers, type 1 (MGI-1); another type (called MGI-2) that induces myeloid differentiation of normal myeloid cells without inducing myeloid cell multiplication; and interleukin-1 (IL-1), which can act on myeloid precursor cells. Different clones of myeloid leukemic cells can differ in their ability to be induced to undergo terminal cell differentiation by different hematopoietic regulatory proteins. We have now studied the ability of cyclosporine A and recombinant IL-1 alpha to regulate in vivo differentiation of different clones of myeloid leukemic cells that are either susceptible or resistant to induction of differentiation by IL-1 in vitro. The results show that (a) cyclosporine A, like other immune- suppressing compounds such as cyclophosphamide, inhibited in vivo differentiation of myeloid leukemic cells and differentiation was restored by injecting recombinant GM-CSF; (b) recombinant IL-1 alpha induced in vivo terminal differentiation of IL-1-sensitive but not IL-1- resistant clones of myeloid leukemic cells; (c) IL-1 alpha and GM-CSF synergistically induced differentiation in vivo in a GM-CSF-responsive and IL-1-nonresponsive clone of leukemic cells; and (d) IL-1 alpha induced in vivo the rapid production and release into serum of the differentiation-inducing protein MGI-2 as well as the growth-inducing proteins M-CSF and G-CSF.     

Def-2, -3, -6 and -8, novel mouse genes differentially expressed in the haemopoietic system

To identify developmentally regulated genes during myeloid differentiation, a self-inactivating retroviral gene-trap vector carrying a beta-galactosidase-neomycin (SA/lacZ/neo) fusion gene was constructed and used to infect myeloid progenitor cells (FDCP-Mix A4). G418-resistant and beta-galactosidase positive cell lines (gene-trap integration [GTI] clones) were established and induced to differentiate in vitro into either macrophages or granulocytes. Expression of the trapped loci was monitored at a single-cell level by analysing the mature cell types for beta-galactosidase activity. All 37 GTI clones tested showed down-regulation either during granulocyte or both granulocytic and macrophage differentiation. The endogenous coding regions fused to the SA/lacZ/neo reporter gene were isolated from eight clones. Molecular analysis revealed that half of them represented novel mouse genes (def-2, -3, -6 and -8) which we confirmed to be differentially expressed in primary haemopoietic tissues. Database searches revealed no significant similarities for def-2 (associated with haemopoietic progenitors) and def-8 (expressed most strongly in peripheral leucocytes). Def-6, which is down-regulated upon the differentiation into myeloid as well as erythroid lineages, was found to be closely related but not identical with the recently described B-cell-specific switch recombinase SWAP-70. Def-3, which is down-regulated upon differentiation into granulocytes but expressed in progenitor cells and macrophages, defines a novel family of RNA binding proteins.     

Constitutive gene expression in myeloid leukemia and cell competence for induction of differentiation by the steroid dexamethasone.

Regulation of the cytoplasmic protein changes during myeloid cell differentiation has been analyzed with two-dimensional gel electrophoresis and differentiation-defective cell mutants. The cells studied include a clone of myeloid leukemia cells (clone 11) that can be induced to differentiate to macrophages by the protein inducer MGI and the steroid dexamethasone (Dex) and mutant clones that were inducible for differentiation to macrophages by MGI but not by Dex. The mutants were not defective in the specific binding of [3H]Dex to cytoplasmic receptors or in the transport and nuclear binding of the receptor--steroid complex. The protein patterns in the mutants showed both specific constitutive protein changes and nonresponding proteins. Twenty-one percent of the Dex-induced protein changes and 2% of the MGI-induced protein changes in clone 11 were constitutively expressed in the mutants. In addition, 28% of the proteins that responded to Dex in clone 11 did not respond to Dex in the mutants, whereas only 4% of the proteins that responded to MGI in clone 11 did not respond to MGI. The higher percentage of constitutive changes was thus associated with a larger defect in induction. The proteins with an abnormal response to Dex still showed a normal response to MGI, and the constitutive changes and nonresponding proteins were different for the two inducers. It is suggested that specific constitutive protein changes expressed by the mutants produced an asynchrony in the developmental program, resulting in a defective response to Dex and to MGI, and that this may apply to other inducers and developmental programs.     

In vivo control of differentiation of myeloid leukemic cells by recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3

The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (colony-stimulating factors [CSF] and interleukin 3 [IL-3], macrophage and granulocyte inducing proteins, type 7 [MGI-1]) and another class (called MGI-2) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemic cells can differ in their response to these regulatory proteins. One type of leukemic clone can be differentiated in vitro to mature cells by incubating with the growth-inducing proteins granulocyte-macrophage (GM) CSF or IL-3, and another type of clone can be differentiated in vitro to mature cells by the differentiation-inducing protein MGI-2. We have now studied the ability of different myeloid regulatory proteins to induce the in vivo differentiation of these different types of mouse myeloid leukemic clones in normal and cyclophosphamide-treated mice. The results show that in both types of mice (a) the in vitro GM-CSF- and IL-3-sensitive leukemic cells were induced to differentiate to mature cells in vivo in mice injected with pure recombinant GM-CSF and IL-3 but not with G-CSF, M-CSF, or MGI-2; (b) the in vitro MGI-2-sensitive leukemic cells differentiated in vivo by injection of MGI-2 and also, presumably indirectly, by GM-CSF and IL-3 but not by M-CSF or G-CSF; (c) in vivo induced differentiation of the leukemic cells was associated with a 20- to 60-fold decrease in the number of blast cells; and (d) all the injected myeloid regulatory proteins stimulated the normal myelopoietic system. Different normal myeloid regulatory proteins can thus induce in vivo terminal differentiation of leukemic cells, and it is suggested that these proteins can have a therapeutic potential for myeloid leukemia in addition to their therapeutic potential in stimulating normal hematopoiesis.