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.     

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.     

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.     

Indirect induction of differentiation in myeloid leukemic cells by lipid A

Normal myeloid and MGI(+)D(+) clones of myeloid leukemic cells can be induced for Fc and complement component 3 rosettes, lysozme, and mature macrophages and granulocytes by a protein with macrophage- and granulocyte-inducing (MGI) activity, whereas MGI(+)D(-) clones can be induced by this protein for rosettes and lysozme but not mature cells. Lipopolysaccharides (LPS) from different bacteria induced the appearance of rosettes, lysozyme, and macrophages in some MGI(+)D(+) clones but did not induce any of these changes in MGI(+)D(-) clones. Lipid A gave the same results as LPS. Incubation of MGI(+)D(+) cells with LPS also induced an MGI activity detectable in the culture medium. This activity behaved like MGI in inducing (i) rosettes, lysozyme, and mature cells in MGI(+)D(+) leukemic cells including a clone resistant to LPS, (ii) rosettes and lysozyme in MGI(+)D(-) leukemic cells, and (iii) differentiation of normal myeloid cells to mature macrophages and granulocytes. This activity was induced in MGI(+)D(+) cells by LPS before the induction of rosettes or lysozyme. The results indicate that the lipid A portion of LPS indirectly induces differentiation of MGI(+)D(+) myeloid leukemic cells by inducing MGI protein. It is suggested that induction of specific regulatory proteins may be a more general mechanism for the induction of differentiation by surface-acting compounds.     

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.     

Concanavalin A receptors on the surface membrane of lymphocytes from patient's with Hodgkin's disease and other malignant lymphomas.

Concanavalin A (Con A) induces movement of its receptors on the cell surface membrane. This induction results in a concentration of Con A site complexes on one pole of the cell to form a cap. A marked difference was found in the mobility of Con A receptor between lymphocytes from normal persons and lymphocytes from patients with Hodgkin's disease and other malignant lymphomas. Lymphocytes isolated from tonsils of patients undergoing tonsillectomy and from axillary lymph nodes of breast cancer patients exhibited approximately 30% of cells with caps, which is identical with the cap formation ability of normal lymphocytes. In biopsy material from patients with Hodgkin's disease and other malignant lymphomas, a significant decrease in the ability of the lymphocytes to form caps was observed. This difference in the mobility of Con A sites was even more pronounced in lymphocytes isolated from the peripheral blood. In 123 patients with Hodgkin's disease and other malignant lymphomas, cap formation ranged between 3 and 12%. The ability of cells, from a normal donor or a lymphoma patient, to form caps was independent of the source from which the lymphocytes were isolated, e.g., lymph node, spleen, or blood. Lymphocytes from patients with lymphoma were also agglutinated by Con A to a higher degree than normal lymphocytes. These findings are discussed in relation to the association of the lymphocytes with these malignancies and as a possible aid in their differential diagnosis.     

Membrane-microtubule interactions: concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins.

The relationship between microtubules and concanavalin A surface receptors during concanavalin A capping in primary cultures of rabbit ovarian granulosa cells was examined by electron microscopic and fluorescence labeling techniques. Cells treated with concanavalin A and hemocyanin at 4 degree or 37 degree and then incubated at 37 degree for 1 hr formed large juxtanuclear caps that were observed with shadow cast replicas of the cell surface. Thin section analysis of capped cells revealed an abundance of microtubules immediately beneath the cap which were arranged approximately perpendicular to the plane of the membrane. The capping process was unaffected by the antimicrotubule agents colchicine or vinblastine. Further, vinblastine treatment of capped calls resulted in the formation of numerous paracrystals that were confined to the cytoplasm underlying the capped region of the membrane; uncapped cells displayed paracrystals that were randomly dispersed in the cytoplasm. Exposure of fixed cells to fluorescein thiocarbamyl colchicine, which localizes colchicine binding proteins, revealed an intensely fluorescent region that corresponded to the cap; this staining pattern was absent in uncapped cells. These findings indicate that concanavalin A mediated capping modifies the cytoplasmic disposition of microtubules and colchicine binding proteins. Further, it is suggested that the capped region of the plasma membrane is a preferred site of microtubule polymerization.     

Morphology, Motility, and Surface Behavior of Lymphocytes Bound to Nylon Fibers

Mouse B lymphocytes that were specifically bound to dinitrophenylated bovine serum albumin on nylon fibers exhibited continuous morphological changes, whereas bound T lymphocytes remained more or less spherical. Cinematomicrographic studies showed that the shape changes were associated with local and global movements, although the attached cells did not translocate along the fiber. Cap formation induced by anti-immunoglobulin was always found to be opposite to the point of attachment. The movements and the shape changes were prevented by cytochalasin B and colchicine. Treatment with these agents did not prevent cap formation but led to randomization of the position of the caps with respect to the fiber. Exposure to concanavalin A or attachment of cells to concanavalin A fibers prevented both movement and patch and cap formation, suggesting that cellular structures regulating the mobility of various receptors are altered by binding to concanavalin A fibers. These observations also indicate that interactions of local areas of the lymphocyte surface with certain ligands and substrates can strongly affect the movement and morphology of the entire cell.     

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.     

Possible role of nucleus-membrane interaction in capping of surface membrane receptors.

Interaction of multivalent ligands and cell surface receptors can induce redistribution of these receptors to form patches and caps. In this study, we have investigated the role of nucleus-membrane interaction in the capping of membrane components. Mouse L cells and leukemia EL4 cells were enucleated with the aid of cytochalasin B, yielding cytoplasts and karyoplasts. Capping of surface receptors was induced by allo- and hetero-immune sera followed by fluorescein-conjugated antiglobulin serum, or by the plant lectin concanavalin A. Capping could easily be induced in intact cells, but virtually no capping was detected in the nucleus-free cytoplasts. Interestingly, karyoplasts, which posses cell-membrane components but very little cytoplasm, could be easily induced to cap their surface antigens. Hence, cap formation of membrane components seems not to be an autonomous membrane process. The data suggest that interaction of surface membranes and inner cell components associated with the nucleus is involved in the movement of surface membrane receptors.     

Ligand-independent cap formation: redistribution of surface receptors on mouse lymphocytes and thymocytes in hypertonic medium.

Most of the mobile receptors on mouse lymphocytes and thymocytes, including immunoglobulins, H-2 antigens, Thy-1.2 antigens, some concanavalin A receptors, and some antigenic determinants detected by anti-thymocyte serum, were redistributed into caps when the cells were incubated in hypertonic medium (about 600 mOsM) in the absence of ligands. The caps reverted to the original distributions if the cells were transferred again to isotonic medium. The viability of the cells was not decreased after incubation in the hypertonic medium. Ligand-independent cap formation appeared to depend upon cellular metabolism. Different species of receptors appeared to move with different mobilities during the process of ligand-independent cap formation. Most microvilli on cells showing caps in hypertonic medium were associated with the regions of the caps. These results suggest that free receptors can be induced to form caps if the receptors are allowed to interact with the machinery of cap formation under special conditions.     

CD8+ minor histocompatibility antigen-specific cytotoxic T lymphocyte clones eliminate human acute myeloid leukemia stem cells

Effective immunotherapy for human leukemia based on infusions of T lymphocytes requires the identification of effector T cells that target the leukemic stem cell. The transplantation of human acute myeloid leukemia into nonobese diabetic/severe combined immune deficient (SCID) mice has identified a rare leukemic progenitor termed the SCID leukemia-initiating cell, which is present in low frequency in the leukemic population and is essential for establishing leukemic hematopoiesis. Thus, this transplant model may be ideally suited to identify effector T cells with antileukemic activity. We report that CD8(+) cytotoxic T lymphocyte (CTL) clones specific for minor histocompatibility antigens inhibit the engraftment of human acute myeloid leukemia cells in nonobese diabetic/SCID mice and demonstrate that this inhibition is mediated by direct CTL recognition of SCID leukemia-initiating cells. These results indicate that CD8(+) minor histocompatibility antigen-specific CTL may be mediators of the graft-versus-leukemia effect associated with allogeneic hematopoietic cell transplantation and provide an experimental model to identify and select T cell clones for immunotherapy to prevent or treat relapse after allogeneic hematopoietic cell transplantation.     

Membrane receptors and their redistribution in lymphoproliferative disorders.

Lymphoid cells from 20 patients with lymphoproliferative disorders, including chronic lymphocytic leukemia, hairy cell leukemia, Sezary syndrome, lymphoma, and lymphadenitis, were studied for redistribution of surface membrane immunoglobulins (SmIg) and concanavalin A (Con-A) receptors. Fluorescein-labeled polyvalent goat anti-human immunoglobulin and fluoresceinated concanavalin A were used as ligands. Results were similar with both ligands. The highest percentage of capping of ligand-membrane receptors was noted in mononuclear cells from patients with "hairy" cell leukemia: from 24% to 90%. These cells showed moderate to marked fluorescein activity and were able to cap within 15 min at 4 degrees C. Chronic lymphocytic leukemia cells showed a weak fluorescein stain with a very low percentage of cells (0%--16%) capping. Lymph node cells from patients with lymphoma demonstrated moderate to strong fluorescein activity with only an average of 3% of the cells capping; while lymphoid cells from patients with lymphaedenitis showed an average of 27.5% capping and moderate fluorescein activity. Capping of Con-A receptors in mononuclear cells from patients with Sezary syndrome was poor (0%--14%) with moderate fluorescein intensity. This report demonstrates difference in density and mobility of binding sites for SmIg and Con-A on the surface membrane of lymphoid cells from various subclasses of lymphoproliferative disorders. These differences may assist in the differential diagnosis and classification of these conditions.     

Lymphocyte receptors for concanavalin A in Hodgkin disease.

The number of lymphocytes with mobile receptors for concanavalin A (Con A) on their surface membrane (forming visible caps after the addition of fluorescein-conjugated Con A) was determined in the peripheral blood of 53 patients with Hodgkin disease. Of 29 individuals studied prior to treatment, the level of capped cells was found to be below the normal range in 9 of 13 in stages I and IIA, 6 of 8 in stage IIIA, and all 8 in stages IIIB and IV. Even among patients in remission 2 yr after successful treatment the level was below the lower normal limit in 9 of 16. The number was also reduced in 7 of 8 individuals with recurrent lymphoma. The level of lymphocytes that cap with Con A may prove to be a more sensitive measure of active Hodgkin disease than the total peripheral lymphocyte count or the level of T cells. This lymphocyte parameter merits further study as a correlate in vitro of cellular immunity.     

Surface phenotypes of human hemopoietic progenitor cells defined by monoclonal antibodies

A panel of ten monoclonal antibodies which react with antigens present on the surface of myeloid leukemic cells was used to investigate the distribution of these antigens on normal hemopoietic stem cells and progenitor cells at various stages of maturity. A population of immature cells, possibly stem cells, that are capable of regenerating CFU-GM in long-term marrow cultures reacts with four antibodies recognizing antigens abundantly expressed in leukemic cells, but does not react with antibodies against Ia-like molecules or against carbohydrate determinants specific for myeloid cells. Progenitor cells that form mixed colonies in semisolid medium (CFU-GEMM), early erythroid (BFU-E) and early myelomonocytic (type 1 CFU-GM) progenitors retain the antigens present on the hypothetical stem cell population and begin to express Ia-like antigens. As they differentiate, myeloid and erythroid progenitors undergo a series of quantitative and qualitative shifts in surface phenotype. They begin to express stage- related, lineage-specific antigens and cease expressing antigens common to early cells of different lineages. The identification of antigens present on very immature normal progenitor cells should be valuable in future studies aimed at the detailed characterization of this relatively little-known hemopoietic cell population.     

Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells

Antibody-targeted chemotherapy is a promising therapy in patients with acute myeloid leukemia (AML). In a phase II study of Mylotarg (CMA-676, gemtuzumab ozogamicin), which consists of a CD33 antibody linked to calicheamicin, saturation and internalization by leukemic and normal myeloid cells were analyzed in 122 patients with relapsed AML. Peripheral blood samples were obtained just before and 3 and 6 hours after the start of the first and second Mylotarg treatment cycles. Within 3 to 6 hours after infusion, near complete saturation of CD33 antigenic sites by Mylotarg was reached for AML blasts, monocytes, and granulocytes, whereas Mylotarg did not bind to lymphocytes. Saturation levels prior to the start of the second Mylotarg treatment cycle were significantly increased compared with background levels before the start of the first cycle. This apparently was caused by remaining circulating Mylotarg from the first treatment cycle (approximately 2 weeks earlier). On binding of Mylotarg to the CD33 antigen, Mylotarg was rapidly internalized, as determined by the decrease in maximal surface membrane Mylotarg binding. Internalization of Mylotarg was also demonstrated in myeloid cells in vitro and was confirmed by confocal laser microscopy. In vitro studies using pulse labeling with Mylotarg showed a continuous renewed membrane expression of CD33 antigens, which can significantly increase the internalization process and thereby the intracellular accumulation of the drug. Finally, Mylotarg induced dose-dependent apoptosis in myeloid cells in vitro. These data indicate that Mylotarg is rapidly and specifically targeted to CD33(+) cells, followed by internalization and subsequent induction of cell death.     

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.     

I and i antigens of human peripheral blood lymphocytes cocap with receptors for concanavalin A.

Surface immunofluorescence experiments using a human anti-i and two anti-I antisera have been performed on human peripheral blood lymphocytes. These are known to contain cold-reactive monoclonal IgM antibodies against the carbohydrate sequence: (formula: see text). A high proportion of B- and T-type lymphocytes express these I and i determinants. In the presence of anti-human immunoglobulin, the cold-reactive membrane-associated complexes of I-anti-I and i-anti-i become stabilized, and redistribution (with patching and capping) can be elicited at 37 degrees C. Dual fluorescence experiments have shown striking concordant staining of I or i (fluorescein) caps and patches with concanavalin A (rhodamine) reactive sites on normal and leukemic cells, suggesting that a proportion of I and i active structures of lymphocyte membranes are structurally associated or physiologically coupled with glycoproteins carrying oligosaccharides with branched mannosyl cores.     

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.