Clones Of Myeloid Leukemic Cells Research Articles

Rescue from programmed cell death in leukemic and normal myeloid cells

Growth factor-independent clones of myeloid leukemic cells can regain a growth factor-dependent state during differentiation. Loss of viability in these differentiating leukemic cells in the absence of growth factor was associated with DNA fragmentation and morphologic changes typical of programmed cell death (apoptosis). The differentiating leukemic cells could be rescued from apoptosis by a hematopoietic growth factor such as interleukin-3 (IL-3) and by the tumor-promoting phorbol ester 12-O-tetra-decanoyl-phorbol-13-acetate (TPA), but not by the nonpromoting phorbol ester 4-alpha-TPA. IL-3 and TPA rescued differentiating myeloid leukemic cells by different pathways and also rescued normal myeloid precursor cells from apoptosis. The rescue of differentiating leukemic and normal myeloid cells by IL-3 or TPA was blocked by amiloride inhibitors of the Na+/H+ antiporter. We suggest that TPA may act as a tumor promoter by inhibiting programmed cell death.

Rescue From Programmed Cell Death in Leukemic and Normal Myeloid Cells

Growth factor-independent clones of myeloid leukemic cells can regain a growth factor-dependent state during differentiation. Loss of viability in these differentiating leukemic cells in the absence of growth factor was associated with DNA fragmentation and morphologic changes typical of programmed cell death (apoptosis). The differentiating leukemic cells could be rescued from apoptosis by a hematopoietic growth factor such as interleukin-3 (IL-3) and by the tumor-promoting phorbol ester 12-O-tetra-decanoyl-phorbol-13-acetate (TPA), but not by the nonpromoting phorbol ester 4-alpha-TPA. IL-3 and TPA rescued differentiating myeloid leukemic cells by different pathways and also rescued normal myeloid precursor cells from apoptosis. The rescue of differentiating leukemic and normal myeloid cells by IL-3 or TPA was blocked by amiloride inhibitors of the Na+/H+ antiporter. We suggest that TPA may act as a tumor promoter by inhibiting programmed cell death.

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.

Selective Regulation of the Activity of Different Hematopoietic Regulatory Proteins by Transforming Growth Factor β1 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.

Indirect induction of differentiation of normal and leukemic myeloid cells by recombinant interleukin 1

Different clones of myeloid leukemic cells can be induced to differentiate to mature macrophages or granulocytes by different normal hematopoietic regulatory proteins. The present experiments with recombinant IL-1α and recombinant IL-1β show that, (a) that there are clones of myeloid leukemic cells which can be induced to differentiate to mature cells by the myeloid cell differentiation-inducing protein MGI-2 and can also be induced to differentiate to mature macrophages and granulocytes by both types of IL-1; (b) this IL-1-induced differentiation is mediated by endogenous production of differentiation-inducing protein MGI-2; (c) IL-1 and MGI-2 induce production of GM-CSF in these leukemic cells; and (d) IL-1 also induces cell differentiation and production of MGI-2 and GM-CSF in normal myeloid precursor cells. The results indicate that IL-1 induces differentiation indirectly.

In Vivo Control of Differentiation of Myeloid Leukemic Cells by Cyclosporine A and Recombinant Interleukin-1α

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.

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.

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.

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.

Target‐cell specificity of hematopoietic regulatory proteins for different clones of myeloid leukemic cells: Two regulators secreted by krebs carcinoma cells

The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (called MGI-1 = CSF or IL-3) and another class (called MGI-2 = DF) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemia cells can differ in their response to these regulatory proteins. The present experiments characterize proteins secreted by Krebs ascites carcinoma cells that induce differentiation of 2 different types of myeloid leukemic cell clones (clones II and 7-M12). The results indicate the following: (1) Krebs cells produce 2 distinct and separable proteins, each inducing differentiation in one of the leukemic clones. (2) One protein induced differentiation of clone-II myeloid leukemic cells and of normal myeloid precursor cells was free of any colony-inducing (MGI-1 = CSF or IL-3) activity, bound to double-stranded mammalian DNA, and was thus a differentiation-inducing protein MGI-2. This MGI-2 protein (MGI-2A) was purified to a single silver-stained band on an SDS polyacrylamide gel. (3) The other protein induced differentiation of clone 7-M12 myeloid leukemic cells, did not bind to double-stranded DNA and could not be separated from the myeloid growth-inducing protein MGI-1GM (GM-CSF) after 6 steps of purification including high-pressure liquid chromatography. The use of specific antisera confirmed that the protein which induced differentiation of clone 7-M12 leukemic cells was MGI-1 GM. The results show that Krebs ascites tumor cells produce 2 different myeloid hematopoietic regulatory proteins that differ in their target specificity for different clones of myeloid leukemic cells.

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.

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.

Role of different normal hematopoietic regulatory proteins in the differentiation of myeloid leukemic cells.

There are 4 different normal myeloid hematopoietic cell growth-inducing proteins MGI-1 (CSF or IL-3) that induce normal precursor cells to multiply and form clones containing only macrophages (MGI-1M = M-CSF = CSF-1), only granulocytes (MGI-1G = G-CSF), both granulocytes and macrophages (MGI-1GM = GM-CSF), or granulocytes, macrophages, eosinophils, mast cells, megakaryocytes and erythroid cells (interleukin-3) (IL-3). There is another type of normal myeloid regulatory protein (MGI-2) with no MGI-1 (CSF or IL-3) activity which can induce differentiation of normal myeloid precursors and certain clones of myeloid leukemic cells. The present results with MGI-2 and pure recombinant MGI-1G, MGI-1GM and IL-3 have shown that different clones of myeloid leukemic cells can be induced to differentiate by different hematopoietic regulatory proteins. One type of leukemic clone is induced to differentiate to mature cells only by MGI-2 and is partially differentiated by MGI-1G, a second type is differentiated only by MGI-1GM or IL-3, and other workers have found a third type that is differentiated only by MGI-1G. The presence of surface receptors does not necessarily make leukemic cells differentiation-competent for these hematopoietic regulatory proteins. All 4 types of MGI-1 (CSF or IL-3) induce endogenous synthesis of MGI-2 in normal myeloid precursor cells. It is suggested that, in addition to their potential therapeutic effect on the development of normal hematopoietic cells, MGI-2, MGI-1G, MGI-1GM and IL-3 all have the potential for differentiation-directed therapy of leukemia in leukemic cells that can be differentiated by one of these normal hematopoietic regulatory proteins.

Regulation of cell-surface receptors for hematopoietic differentiation-inducing protein MGI-2 on normal and leukemic myeloid cells.

The normal myeloid hematopoietic regulatory proteins include 4 different growth-inducing proteins (IL-3, MGI-1GM = GM-CSF, MGI-1G = G-CSF, and MGI-1M = M-CSF = CSF-1). There is also another type of normal myeloid regulatory protein (MGI-2) with no MGI-1 (CSF or IL-3) activity, which can induce differentiation of normal myeloid precursors and certain clones of myeloid leukemic cells. Studies on the binding of MGI-2 to differentiation-competent (D+) and differentiation-defective (D-) clones of mouse myeloid leukemic cells and to normal cells indicate that: (1) D+ clones of myeloid leukemic cells had about 2,500 high-affinity surface receptors per cell, like mature normal myeloid cells, and the bound MGI-2 was rapidly internalized with its cell-surface receptors at 37 degrees C causing down-regulation of MGI-2 receptors in both the normal and leukemic cells; (2) in some D- clones, the number and internalization of MGI-2 receptors were similar to those of D+ clones whereas other D- clones had only 0-100 MGI-2 receptors per cell; (3) normal thymus and lymph-node lymphocytes and T lymphoma cells did not show detectable MGI-2 receptors; (4) there was an independent expression of receptors for MGI-2 and for the 4 myeloid growth-inducing proteins on different clones of myeloid leukemic cells; and (5) none of the 4 myeloid growth-inducing proteins IL-3, MGI-1GM, MGI-1G, or MGI-1M, inhibited binding of MGI-2 to its receptors. The cytotoxic proteins lymphotoxin and tumor necrosis factor did not induce differentiation of the mouse myeloid leukemic cells and also did not inhibit binding of MGI-2 to its receptors. These results show that the myeloid differentiation-inducing protein MGI-2 binds to cell-surface receptors that are different from the receptors for the 4 myeloid growth-inducing proteins and these cytotoxic proteins.

Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells.

There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M-CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells.

Regulation of in-vivo differentiation of myeloid leukemic cells by antigen-specific helper T lymphocytes

There are clones of myeloid leukemic cells that can be induced to differentiate in vitro and in vivo by normal macrophage and granulocyte differentiation-inducing protein MGI-2 ( = DF). The differentiation of these myeloid leukemic cells in vivo is regulated by a cell mediated immune response which requires T lymphocytes. We now show that differentiation of myeloid cells in vivo can be induced by antigen-specific helper T lymphocytes and that this is associated with the ability of the helper T cells to produce myeloid cell differentiation-inducing protein MGI-2. Antigen specific helper T cells can accumulate at a site that contains the antigen. It is suggested that migration in response to antigen of helper T cells producing differentiation factors may play an important role in inducing in vivo differentiation of leukemic cells.

Control of in-vivo differentiation of myeloid leukemic cells— V. Regulation by response to antigen

Regulation of in-vivo differentiation of myeloid leukemic cells by response to antigen was analysed with different clones of mouse myeloid leukemic cells and human myeloid leukemic cells (HL-60). Differentiation was studied in diffusion chambers implanted into the peritoneal cavity of mice and the antigens used were bovine serum albumin and chicken ovalbumin. It is shown that the presence of either of these antigens in the diffusion chambers can induce differentiation in MGI + D + mouse and human myeloid leukemic cells, and that pre-immunization with antigen enhanced this in-vivo differentiation. This enhancement showed immunological specificity and was transferred from immunized to non-immunized mice by spleen cells enriched for T lymphocytes. In contrast to these results with MGI + D + clones of myeloid leukemic cells, clones of WEH1-3B myeloid leukemic cells were induced to differentiate in vivo to the same extent either in the presence or absence of antigen. The results indicate: (1) that in-vivo differentiation of MGI + D + clones of myeloid leukemic cells can be induced by response to antigen and (2) that in vivo differentiation of different clones of myeloid leukemic cells can be regulated in different ways.

Control of in vivo differentiation of myeloid leukemic cells. IV. Inhibition of leukemia development by myeloid differentiation-inducing protein.

It is shown that a 5-day schedule of two injections per day of the myeloid differentiation-inducing protein MGI-2 inhibited the in vivo development of leukemia in SL and SJL/J mice with different syngeneic MGI+D+ clones of myeloid leukemic cells. With this schedule of treatment high levels of MGI-2 were maintained in the serum for long periods. In contrast to these results with MGI-2, the same schedule of injections of the myeloid growth-inducing protein MGI-I did not affect the in vivo development of leukemia in mice with MGI+D+ myeloid leukemic cells, but stimulated normal myelopoiesis in the bone marrow. Different forms of MGI-I including MGI-IM and MGI-IG had different serum half-lives, and the form of MGI-I with the shortest serum half-life showed the smallest in vivo effect on normal myelopoiesis. MGI-2 injections did not inhibit the in vivo development of differentiation-defective WEHI-3B myelomonocytic leukemic cells or YAC lymphoma cells. The results indicate that the in vivo inhibitory effect of MGI-2 on the development of myeloid leukemia correlated with its differentiation-inducing potential on the leukemic cells. It is concluded that this approach of inhibiting leukemia development by inducing differentiation should also be applied to human leukemic patients, whose cells have been shown to be inducible for differentiation in culture by human MGI-2 or by other differentiation-inducing compounds.

Coupling of growth and differentiation in normal myeloid precursors and the breakdown of this coupling in leukemia.

Normal myeloid precursors are dependent on the macrophage and granulocyte growth-inducing protein MGI-1 for cell viability and multiplication. MGI-1 also induces production of the differentiation-inducing protein MGI-2, and this induction of a differentiation-inducing protein by a growth-inducing protein provides a mechanism for the normal coupling of growth and differentiation. It is shown that this induction of MGI-2 by MGI-1 occurs in the myeloid precursors and not in some other cells in the normal bone marrow, that the induced MGI-2 can be detected 6 h after the addition of MGI-1, and that MGI-2 can be induced in these cells by purified MGI-1. There are clones of myeloid leukemic cells that no longer require MGI-1 for cell viability and multiplication, but in which this requirement for MGI-1 can be restored after induction of differentiation by MGI-2. A similar concentration of MGI-1 was required for the optimum induction of growth in these differentiating leukemic cells and in normal myeloid precursors. In the presence of MGI-1 these differentiating leukemic cells multiplied and then lost their differentiation-associated properties. In contrast to normal myeloid cells, MGI-1 did not induce MGI-2 in the MGI-1 requiring differentiating myeloid leukemic cells. This lack of induction of MGI-2 by MGI-1 occurred in cells cultured in serum-containing or serum-free-medium, and can explain the loss of differentiation-associated properties. The results indicate that there has been a genetic breakdown of the normal coupling mechanism between growth and differentiation in these leukemic cells so that MGI-1 can no longer induce MGI-2.

Cell competence for industion of differentiation by insulin and other compounds in myeloid leukemic clones continuously cultured in serum- free medium

Clones of myeloid leukemic cells varying in their competence for induction of differentiation have been continuously grown in serum-free medium. In the medium used, which contained transferrin, the growth rates of these cells were nearly similar to those found in serum- containing medium. The clones also maintained in this medium their competence for induction of differentiation by the normal macrophage and granulocyte differentiation-induction protein MGI-2, the steroid dexamethasone, and lipopolysaccharide. In contrast to the results with these inducters, some clones continuously cultured in a serum-free medium showed a gain of inducibility by insulin and another clone a gain of inducibility by the tumor promoter 12-O-tetradecanoylphorbol-13- acetate in low serum and serum-free medium. Induction of differentiation by these two compounds was therefore inhibited in these clones by the presence of serum. It is suggested that serum-free medium may also show the existence of other inducers of differentiation not detected in serum-containing medium and that these results are relevant to the possible therapeutic use of compounds such as insulin for the induction of normal differentiation in leukemic cells in vivo.