In vivo inhibition of the development of myeloid leukemia by injection of macrophage-and granulocyte-inducing protein

It is shown that injection of macrophage- and granulocyte-inducing protein (MGI) can inhibit the development of myeloid leukemia in vivo and stimulate normal myelopoiesis. The MGI injected contained MGI-1 activity that induces colony formation with normal myeloblasts, and MGI-2 activity that induces differentiation of normal and MGI⁺D⁺ leukemic myeloblasts to mature macrophages or granulocytes. The MGI preparations injected did not contain interferon and it was shown that the results obtained were not due to minute amounts of contaminating lipopolysaccharide (LPS). Intraperitoneal injection of MGI stimulated myelopoisesis in normal adult mice. Markedly higher levels of serum MGI activity could be obtained by injecting MGI than by injecting LPS, a compound that stimulates the in vivo production of MGI. Injection of MGI into mice that had been inoculated intravenously with MGI⁺D⁺ or MGI⁺D^? myeloid leukemic cells showed that, 3 weeks after the beginning of treatment, there was a 3- to 5-fold decrease in the number of leukemic colony-forming cells and an increase in mature granulocytes in the bone marrow, together with a 2- to 5-fold decrease in the percentage of morphologically identified myeloid blast cells in the bone marrow and peripheral blood. The remaining leukemic colony-forming cells in the MGI-treated mice inoculated with MGI⁺D⁺ cells were not resistant to the induction of differentiation by MGI-2. Injection of LPS to mice inoculated with LPS-resistant MGI⁺D^? leukemic cells also inhibited the development of leukemia, but this inhibition was less effective than with MGI and was presumably indirect. Injection of MGI reduced the tumor volume in mice subcutaneously inoculated with leukemic cells at least 50-fold. The median survival time of MGI-treated mice inoculated intravenously with MGI⁺D⁺ cells or subcutaneously with MGI⁺D^? cells was increased by about 40%, and 50% of the MGI-treated mice inoculated subcutaneously with MGI⁺D⁺ cells did not develop tumors. MGI also increased the anti-tumor effect of cyclophosphamide. The leukemia-inhibiting activity in the injected MGI preparations was associated with the peak of MGI activity separated on a hydroxylapatite column and was destroyed by treatment at the temperature which destroys MGI-2 activity. This indicates that the inhibition of leukemia development was mediated by MGI-2. It is suggested that improved schedules of MGI treatment, with or without compounds used in the cytotoxic types of therapy, should be able to give an even better inhibition of leukemia development and that these results should also be applicable to the therapy of human myeloid leukemia.     

Control of in vivo differentiation of myeloid leukemic cells. III. Regulation By T Lymphocytes And Inflammation

Mouse and human (HL-60) MGI⁺D⁺ myeloid leukemic cells were induced to differentiate to mature cells in diffusion chambers implanted into the peritoneal cavity of normal mice when a xenogeneic source of serum was added to the diffusion chambers. Differentiation was inhibited in immune deficient mice including congenitally athymic nude and neonatally thymectomized mice, and mice treated with cyclophosphamide, hydrocortisone, or X-irradiation. There was no such inhibition of differentiation in mice with various genetic defects in their B lymphocytes, granulocytes, erythrocytes and natural killer cells. Differentiation in cyclophosphamide-treated mice was restored by a single intravenous injection of normal spleen cells highly enriched for T lymphocytes. Conditions permissive for differentiation were associated with a higher number of eosinophils in the peritoneum than conditions that inhibited differentiation. Intraperitoneal injections of inflammatory peritoneal exudate cells, peritoneal granulocytes, or the inflammation inducer sodium caseinate, restored the ability of defective mice to induce differentiation. Injections into defective mice of the normal mouse macrophage and granulocyte differentiation-inducing protein (MGI-2) restored differentiation of the mouse myeloid leukemic cells but not of the human myeloid leukemic cells. Differentiation of normal mouse bone marrow myeloid precursors to mature cells and of differentiation-defective (MGI-D-) mouse myeloid leukemic cells to intermediate stages of differentiation were not affected by the conditions that inhibited differentiation of the MGI ⁺D⁺ myeloid leukemic cells. The results indicate: 1) that the intraperitoneal accumulation of inflammatory cells, including eosinophils, can induce differentiation of MGI ⁺D⁺ leukemic cells in the peritoneal cavity; 2) that this response requires T lymphocytes and can be regulated by xenogeneic serum in the chamber; 3) that in vivo diffentiation of normal and MGI ⁺D⁺ myeloid leukemic cells can be regulated in different ways; and 4) that the in vivo differentiation of the mouse MGI ⁺D⁺ leukemic cells, human MGI⁺D⁺ leukemic cells and mouse MGI^?D^? leukemic cells were induced by different compounds, so that differentiation of different types of leukemic cells may be differently regulated in vivo depending on which compounds induce differentiation.     

Regulation and role of different macrophage- and granulocyte-inducing proteins in normal and leukemic myeloid cells

It has previously been shown that there are different molecular forms of macrophage- and granulocyte-inducing (MGI) proteins; one form, MGI-I, induced the formation of colonies with differentiated cells from normal myeloblasts and another form, MGI-2, induced normal differentiation in MGI⁺D⁺ leukemic myeloblasts that no longer require MGI-I to form colonies. The present results indicate that MGI-2 can also induce differentiation (without inducing colony formation) in the normal cells, and that MGI-I induced MIG-2 in the normal but not in the leukemic cells. It is suggested from these results that MGI-2 is the differentiation-inducing protein for normal and leukemic cells whereas MGI-I is the growth-inducing protein that induces colony formation by the normal cells, and that induction of differentiation in the normal cell colonies is due to induction of MGI-2 by MGI-I.     

Clonal regulation of the induction of macrophageand granulocyte-inducing proteins for normal and leukemic myeloid cells

A cloned line of myeloid leukemic cells can be induced by the alkylating agent nitrosoguanidine for two macrophage- and granulocyte-inducing (MGI) activities. One activity, MGI-1, induced the formation of macrophage and granulocyte colonies from normal myeloblasts. Another activity, MGI-2, induced differentiation of MGI⁺D⁺ myeloid leukemic cells to macrophages and granulocytes. Experiments on the time course of induction of the two activities have shown that MGI-1 was induced before MGI-2. MGI-1 was first detected in cell extracts and this was followed by detection of both activities in culture supernatants (conditioned medium). After induction with bacterial lipopolysaccharide, another inducer of both MGI activities in this clone, MGI-1 was also detected before MGI-2 in cell extracts. The steroid dexamethasone, which is an effective inducer of some differentiation-associated properties in this clone, did not induce either MGI-1 or MGI-2. Studies with different clones of myeloid leukemic cells have shown a clonal variation in the induction of MGI-1 and MGI-2. Different clones were induced by nitrosoguanidine either for MGI-1 and MGI-2, for MGI-1 without MGI-2, or for neither MGI-1 nor MGI-2. None of the clones were induced for MGI-2 without MGI-1. The results indicate that the induction of MGI-1 and MGI-2 is differently regulated in the same clone, and that there is a clonal and thus presumably genetic variation in inducibility for these two activities of MGI.     

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-1 alpha and recombinant IL-1 beta 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.     

Genetic dissociation of different cellular effects of interferon on myeloid leukemic cells.

Mutant clones of mouse myeloid leukemic cells have been used to study the effect of mouse interferon on the expression of different cellular genes. The cell mutants, MGI⁺D⁺, MGI⁺D^? and MGI^?D^?, differed in their competence to be induced to undergo cell differentiation by the normal macrophage and granulocyte protein inducer MGI or the steroid hormone dexamethasone. The growth of all the clones tested was inhibited by interferon, but there was a 20-fold difference in sensitivity to growth inhibition in different clones. This difference was not associated with the same difference in susceptibility to the cytotoxic effect of cytosine arabinoside and actinomycin D, or with cell competence for the induction of differentiation. The variation in cytotoxic effect of interferon on different malignant cell populations should, therefore, be taken into consideration in the possible clinical use of interferon as an anti-tumor agent. Some MGI⁺D⁺ clones, but none of the MGI⁺D^? or MGI^?D^? clones, showed, in addition to a high sensitivity to growth inhibition by interferon, a 14- and 2-fold enhancement of the induction of lysozyme by dexamethasone and MGI, respectively, and a slight induction of Fc rosettes with dexamethasone. But interferon had no effect on induction of C3 rosettes, immune phagocytosis and differentiation to mature macrophages or granulocytes. The enhancing effect on lysozyme induction by interferon was also obtained with cordycepin, vinblastine, or 2-deoxy-D-glucose, although neither interferon nor any of these other compounds alone induced any of the differentiation-associated properties. A MGI⁺D^? mutant, derived from an MGI⁺D⁺ clone with an enhancement of lysozyme induction by interferon, showed the same high sensitivity to growth inhibition by interferon as its parental MGI⁺D⁺ clone, but there was no enhancement of the induction of lysozyme by MGI or dexamethasone. The results indicate that interferon can exert a selective enhancing effect on the expression of some genes involved in differentiation in clones with the appropriate genotype and that, by using appropriate cell mutants, it is possible to genetically dissociate different cellular effects of interferon.     

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-I for cell viability and multiplication. MGI-I 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-I 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-I, and that MGI-2 can be induced in these cells by purified MGI-I. There are clones of myeloid leukemic cells that no longer require MGI-I for cell viability and multiplication, but in which this requirement for MGI-I can be restored after induction of differentiation by MGI-2. A similar concentration of MGI-I was required for the optimum induction of growth in these differentiating leukemic cells and in normal myeloid precursors. In the presence of MGI-I these differentiating leukemic cells multiplied and then lost their differentiation-associated properties. In contrast to normal myeloid cells, MGI-I did not induce MGI-2 in the MGI-I requiring differentiating myeloid leukemic cells. This lack of induction of MGI-2 by MGI-I 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-I can no longer induce MGI-2.     

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.     

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 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.     

Clonal variation in susceptibility to differentiation by different protein inducers in the myeloid leukemia cell line M1

Differentiation-competent clones of myeloid leukemic cells, independently isolated from the M1 cell line in Rehovot, Israel, and in Saitama, Japan, can be induced to differentiate to mature cells by the protein which we called macrophage and granulocyte differentiation-inducing protein-2 (MGI-2) that we have shown is interleukin 6 (IL-6). We now show that our MGI-2/IL-6-susceptible clones of M1 cells were not induced to differentiate with the differentiation-inducing protein called D-factor/leukemia inhibitory factor (LIF) which has also been called human interleukin for DA cells (HILDA), whereas this protein induced differentiation to macrophages in the M1 clone isolated in Saitama which was also used in Melbourne, Australia, The D-factor/LIF susceptible clone also showed a 4-fold lower sensitivity to MGI-2/IL-6 than the D-factor/LIF resistant clone. Both types of clones differentiated with interleukin-1 alpha (IL-1 alpha) and dexamethasone, whereas the D-factor/LIF resistant clone, but not the D-factor/LIF susceptible clone, was induced by bacterial lipopolysaccharide (LPS) to differentiate to mature macrophages. The present results show that clonal differences in susceptibility to differentiation-inducing proteins in the M1 cell line can explain the isolation of different differentiation-inducing proteins in M1 leukemic cells in different laboratories.     

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.     

Control of normal differentiation of myeloid leukemic cells. V. Normal differentiation in aneuploid leukemic cells and the chromosome banding pattern of D+ and D minus clones

A line of mouse myeloid leukemic cells in culture contained two types of clones. One type can be induced by the differentiation-inducing protein MGI to undergo normal differentiation to mature macrophages and granulocytes (D⁺ clones), whereas the other type could not be induced to differentiate (D^? clones). D⁺ clones can segregate some D^? progeny and D^? clones can segregate some D⁺ progeny. The segregant clones were more unstable in their ability to differentiate, than the stable parental clones from which they were derived. The chromosome banding pattern has been analyzed in 10 stable and six unstable clones. The clones had a modal chromosome number of 39, 40 or 41, and none of the clones, not even those with 40 chromosomes, showed a normal diploid banding pattern. Stable D⁺ and D^? clones had different karyotypes. However, the banding patterns of the unstable D⁺ and D^? segregants were not detectably different from the parental clones from which they were derived. The results indicate that normal differentiation can occur in aneuploid myeloid leukemic cells; that the initial segregation from D⁺ to D^? and from D^? to D⁺ was due to a hereditary change which did not show chromosome changes detectable by the banding technique; and that detectable chromosome changes were associated with hereditary stabilization of the difference between D⁺ and D^?.     

Separation of different molecular forms of macrophage- and granulocyte-inducing proteins for normal and leukemic myeloid cells

It is shown that serum of mice treated with endotoxin (ES) contains three separable and functionally distinct forms of macrophage- and granulocyte-inducing (MGI) proteins. One form (MGI-1M) induced the formation of macrophage colonies from normal bone-marrow cells and showed on gel filtration an apparent molecular weight of 300,000; a second form (MGI-1G) induced the formation of granulocyte colonies from normal bone-marrow cells and had an apparent molecular weight of 45-100,000; and the third form (MGI-2) induced the normal differentiation of MGI+D+ myeloid leukemic cells to macrophages and granulocytes and had an apparent molecular weight of 28,000. Studies on the time course of the decrease of these three activities in ES have indicated that MGI-2 was more readily inactivated in vivo than MGI-1M and MGI-1G. The MGI-1M in ES isolated after gel filtration was completely neutralized by an antiserum to MGI-1 from mouse L-cells, whereas the isolated MGI-1G and MGI-2 were not affected by this antiserum. Gel filtration under dissociating conditions (6 M guanidinium chloride) resulted in a reduction of the apparent molecular weights of MGI-1M from 300,000 to 42,000, and of MGI-1G from 45-100,000 to 28,000, while it produced no change in the 28,000 apparent molecular weight of MGI-2. Similar studies with conditioned medium produced in vitro from mouse lung and peritoneal macrophages showed that in these conditioned media, MGI-1 (both G and M) in the native form had an apparent molecular weight of 41,000 and MGI-2 of 24,000, and that both MGI-1 and 2 had an apparent molecular weight of 24,000 under dissociating conditions. The results indicate that MGI-1 exists in serum in vivo and in these conditioned media as aggregated proteins, whereas MGI-2 does not, and that macrophages and lung tissue are not the only source of the MGI proteins found in ES. It is suggested that all three forms of MGI activity are derived from one precursor protein; that only the MGI-2 form assayed on leukemic cells should be used for treatment based on the induction of normal cell differentiation in myeloid leukemia; and that MGI-2 may serve as a survey mechanism for inducing differentiation in myeloid leukemic cells that have lost their responsiveness to the MGI-1 molecules that control the viability, proliferation and differentiation of normal myeloblasts.     

Independent regulation of myeloid cell growth and differentiation inducing proteins: in vivo regulation by compounds that induce inflammation

Regulation of the in vivo production of myeloid cell growth-inducing (MGI-1) and differentiation-inducing (MGI-2) proteins has been studied in mice injected with the inflammation-inducing compounds sodium caseinate, thioglycollate and bacterial lipopolysaccharide. The results indicate that these inflammation-inducing compounds can induce in vivo production of MGI-1 and MGI-2; that different inducing agents can cause a different body-distribution of MGI-1 and MGI-2; that there is an independent regulation of in vivo production and distribution of MGI-1 and MGI-2; and that there is a granulocyte growth-inducing protein (MGI-IG = G-CSF) that is not identical to the differentiation-inducing protein (MGI-2). Resident peritoneal macrophages produce MGI-1 and MGI-2 in vitro, but inflammatory macrophages show a reduced ability to spontaneously produce these proteins after in vivo injection of caseinate or thioglycollate. The results thus also indicate that macrophage activation can affect the ability of macrophages to produce the myeloid cell regulatory proteins MGI-1 and MGI-2.     

Induction of specific changes in the surface membrane of myeloid leukemic cells by steroid hormones

Normal mature macrophages and granulocytes have surface membrane receptors for specific immunoglobulin and immunoglobulin complement, which can be detected by rosette formation with erythrocytes coated with antibody (EA) or with antibody and complement (EAC). There are three types of myeloid leukemic cells, IR⁺D⁺, IR⁺D^? and IR^?D^?. IR⁺D⁺ cells were induced to form receptors for EAC but not for EA by the steroid hormones prednisolone, dexamethasone and estradiol. Induction required protein synthesis and was not inhibited by cordycepin or vinblastine. Optimum induction required the continued presence of the hormones. IR⁺D⁺ cells were also induced by these hormones to migrate in agar, attach to the surface of a Petri dish and form macrophages. IR⁺D^? cells showed a lower inducibility by these hormones and no formation of macrophages. There was no induction of any of these changes with IR^?D^? cells. The steroid hormones progesterone, testosterone and cortisone did not induce these changes in any of the leukemic cells and inhibited induction by prednisolone, dexamethasone and estradiol. The results indicate that specific surface membrane changes in myeloid leukemic cells can be induced by certain steroid hormones.     

Screening for induction of differentiation and toxicity to blast cells by chemotherapeutic compounds in human myeloid leukemia

Bone marrow cells from 9 patients with acute myeloid leukemia and 1 patient with a blast crisis of chronic myeloid leukemia were cultured to determine their ability to be induced to differentiate by different chemotherapeutic compounds. Five of these 10 patients showed differentiation to granulocytic and/or monocytic cells by culture with medium containing the myeloid cell differentiation-inducing protein MGI-2. Actinomycin D induced differentiation in cells from 2 of the patients who did not show differentiation with MGI-2 containing medium. In these 7 patients there was an increase in the ratio of differentiated myeloid cells to blasts. None of these 10 patients showed induction of differentiation by cytosine arabinoside, adriamycin, or daunomycin, but treatment with these compounds showed in some patients an increase in the ratio of differentiated myeloid cells to blasts. The results indicate that this ratio can be increased by differentiation and also in some patients by toxicity to blast cells. With dexamethasone or vinblastine there was no induction of differentiation and no increase in this ratio in any of the 10 patients tested. After in vivo chemotherapy with low dose cytosine arabinoside, cells from one patient showed a similar response in culture to actinomycin D as cells before chemotherapy, whereas in another patient the cells had acquired the ability to respond to actinomycin D. In contrast, after high-dose in vivo chemotherapy with cytosine arabinoside and daunomycin, cells from a third patient seemed to have lost the ability to differentiate in vitro by MGI-2 containing medium or actinomycin D. The results indicate that pre-screening for differentiation-inducing compounds and compounds that show toxicity to blast cells should be useful to select the appropriate compounds to be used for therapy, and that it is advisable to screen the cells both before and after initiation of therapy.     

Cytogenetic analysis of human acute and chronic myeloid leukemic cells cloned in agar culture

Karyotypic analysis was performed on agar cultures of blood or bone marrow from 12 patients with acute or chronic myeloid or myelomonocytic leukemia in whom karyotypic markers were present. The granulocytic colonies and clusters which developed on culture were shown to be derived from representative cells of the leukemic population. In two patients with acute leukemia in remission, normal colonies with a normal karyotype were grown from marrow cells but in two patients with chronic myeloid leukemia in remission the Ph¹ abnormality persisted in colony cells. The agar culture technique appears to be ideal for following the emergence and disappearance of leukemic and normal granulopoietic populations in patients with these types of leukemia.     

Autoinduction of differentiation in wehi-3B leukemia cells

Cells of the differentiation-responsive mouse myelomonocytic leukemia cell line WEHI-3B D⁺ form colonies in agar exhibiting a low frequency of spontaneous differentiation mainly in the macrophage pathway. Compared with undifferentiated colonies, spontaneously differentiating colonies have a reduced content of clonogenic cells and surviving clonogenic cells tend themselves to form differentiating colonies, both being characteristics of differentiated colonies induced by the regulator, granulocyte colony-stimulating factor, G-CSF. Colony crowding increased the frequency of spontaneously differentiating colonies and WEHI-3B D⁺ colony cells were shown to release material able to induce differentiation in WEHI-3B D⁺ colonies. Cells from spontaneously differentiating D⁺ colonies were not hyperresponsive to the induction of differentiation by G-CSF and did not release larger amounts of differentiation-inducing material than did cells from undifferentiated colonies. Cells of the differentiation-unresponsive WEHI-3B D^? line produced similar amounts of differentiation-inducing material to those produced by D⁺ cells. Apparently spontaneous differentiation in WEHI-3B D⁺ colonies seems most likely to be due to exposure of the colony-forming cell or its ancestors to a differentiation-inducing factor of WEHI-3B origin prior to culture in agar, the genetic program initiating differentiation being inherited by the progeny of the exposed cell.     

The Fc‐alpha receptor is a new target antigen for immunotherapy of myeloid leukemia

Antibody‐based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64‐targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti‐human CD89 single‐chain Fragment variable and the well‐characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC₅₀ values in range 0.2–3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose‐dependent apoptosis‐mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89‐specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89‐ETA' as a potent, novel immunotherapeutic agent.