Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.     

The effects of recombinant CSF-1 on the blast cells of acute myeloblastic leukemia in suspension culture

Recombinant hemopoietic colony-stimulating factors (CSFs), including GM-CSF, G-CSF and IL-3, have been shown to be effective stimulators of both self-renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF-1 (or M-CSF), on the growth of leukemic blasts in culture. CSF-1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF-1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF-1 may be useful in the treatment of selected cases of AML.     

Ex vivo expansions of megakaryocytopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free culture supplemented with proteoglycans extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of whale

As a possible approach to the treatment of thrombopocytopenia, the ex vivo expansion of megakaryocytic progenitor cells may be a useful tool to accelerate platelet recovery in vivo. Our objective was to assess the promoting effect of proteoglycans in a serum-free culture condition using human cord blood CD34(+) cells. Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of a whale were applied to the ex vivo expansion of megakaryocytopoiesis and thrombopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free cultures stimulated with a combination of thrombopoietin (TPO) and interleukin-3 (IL-3). Each PG (0.5 and 5 mug) was applied to the culture with three different concentrations of TPO (50, 5 and 0.5 ng/ml) and IL-3 (100, 10 and 1 ng/ml). Both of the PGs showed no promoting effects on the mononuclear cell proliferation rate in any of the cultures. However, the whale-PG promoted the generation of megakaryocytic progenitor cells and megakaryocytes in the culture with a lower dose of cytokines, respectively. In addition, whale-PG led to a significant increase in CD42a(+) particles which seemed to be platelets. While the salmon-PG failed to promote such production in almost all of the cultures. Although whale-PG is an attractive molecule for the ex vivo expansion of human megakaryocytopoiesis, its action may depend on the glycosaminoglycans sulfation pattern and the ability of the binding affinity and the kinetics to interact with the cytokines and hematopoietic stem/progenitor cells.     

New human myelodysplastic cell line,TER-3: G-CSF specific downregulation of Ca2+/calmodulin-dependent protein kinase IV

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS.     

Identification of monocytic nature in acute undifferentiated leukemia by in vitro marrow culture study

We report a case with acute undifferentiated leukemia whose leukemic blasts lacked morphological, cytochemical and immunological features of lymphoid or myeloid differentiation. The in vitro culture study defined her leukemia as of monocytic origin. Her marrow blasts underwent monocytic differentiation with strong nonspecific esterase activity when cultured in a liquid system with human placental conditioned medium. The semisolid agar culture showed an AML-type growth pattern. The present study indicates that in vitro culture study can be used as a supplement to improve the classification of certain unclassifiable leukemias.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors

Interleukin-3 (IL-3)-dependent cell lines (FDCP-mix) were cloned and isolated from long-term bone-marrow cultures infected with src-MoMuLV. These cell lines have many of the characteristics of hematopoietic stem cells. Early isolates of the FDCP-mix cells form spleen colonies in irradiated mice and establish long-term hematopoiesis on irradiated marrow stroma in vitro in the absence of IL-3. These two properties of the cells are lost within 15 weeks of establishing the cell lines, but the cell lines retain their ability to differentiate in a multilineage response to hematopoietic growth factors and to hematopoietic stromal cells, as well as to self-renew in the presence of IL-3. The choice between differentiation and self-renewal in FDCP-mix cells can clearly be modified by culture conditions: in particular, cultures containing horse serum preferentially promote self-renewal, whereas cultures containing fetal calf serum preferentially promote differentiation. The FDCP-mix cell lines are not leukemic, nor do they contain the src oncogene. Their ability to respond to hematopoietic growth factors and stroma in a similar manner to normal hematopoietic cells makes them a valuable model for studying the regulation of hemopoietic cell self-renewal and differentiation.     

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.     

Autocrine regulation of terminal differentiation by interleukin-6 in the pluripotent KU812 cell line

The human KU812 leukemic cell line is a model for studying cell commitment towards different hematopoietic lineages. Indeed, this cell line is characterized by both a capacity for self-renewal and the ability to differentiate spontaneously along erythroid and basophilic cell lineages. In this study we show that interleukin-6 (IL-6) and its specific receptor (IL-6-R) are spontaneously expressed in the human KU812 cell line. Addition of antibody against IL-6 weakly inhibited its cell proliferation (20 to 30%) suggesting that the endogenous production of IL-6 was partially responsible for the growth of the cell line. In contrast, the spontaneous terminal differentiation of this cell line towards the erythroid and basophilic lineages was inhibited by an antibody against IL-6 and this effect was reversed by addition of recombinant human IL-6 (rIL-6). These results suggest that IL-6 is involved more in differentiation than in the proliferation of KU812 cells. After several passages, KU812 cells lose their capacity to differentiate spontaneously. In these cells, the IL-6-R was no more detectable. We therefore suggest that this loss of spontaneous differentiation is associated with an interruption of the IL-6 autocrine loop.     

Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6

Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45(+) and CD11b(+) cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin(-) Sca-1(+)c-Kit(+) (LSK) hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion.     

Regulation of human megakaryocytopoiesis: analysis of proliferation, ploidy and maturation in liquid cultures

A liquid culture technique associated with either double staining and flow cytometry or electron microscopy was used to study human megakaryocytopoiesis. During development from the embryo to the adult, a progressive increase in ploidy classes associated with an enhancement of megakaryocyte (meg) size was observed. Granulocyte-macrophage colony-stimulating factor had no effects on adult marrow cultures. In contrast, interleukin (IL) 3 induced a marked proliferation, but was unable to promote polyploidization. Furthermore, it abrogated the effects on endomitosis of aplastic plasma (AP). This negative effect on polyploidization of IL-3 could be partially dissociated from its effects on proliferation by a delayed addition in culture. AP acted on both proliferation and endoreplication, which was not due to the main hematopoietic growth factors, including IL-6. A synthesis of IL-6 was detected by in situ hybridization in cultured cells including megs which also express receptors for IL-6. These results suggest that terminal meg differentiation may be regulated by an autocrine IL-6 loop, and that megakaryocytopoiesis may be independently regulated at early and late stages of differentiation.     

Detection and characterization of a B cell stimulatory factor (BSF-TC) derived from a bone marrow stromal cell line

Stromal cell lines derived from murine bone marrow support the growth of immature pre-B cells and produce cytokines that affect the growth and differentiation of other hematopoietic precursors. Conditioned medium (CM) from one such line (TC-1) stimulated marked proliferation of B cells previously activated by anti-Ig (anti-Ig blasts). Proliferation of anti-Ig blasts was not induced by purified cytokines known to be produced by TC-1 (CSF-1, GM-CSF, or G-CSF) or by IL-1, IL-2, IL-3, IL-4, IL-5, or IL-6. Furthermore, IL-2, IL-4, and IL-5, alone or in combination, failed to support proliferation or differentiation of anti-Ig blasts. TC-1 CM enhanced proliferation of B cells that were co-cultured with LPS, anti-Ig, or dextran sulfate; co-stimulation with anti-Ig was unaffected by the presence of monoclonal anti-IL-4. Proliferation of low, but not high, density B cells isolated from spleen was directly stimulated by TC-1 CM. These results suggest that bone marrow stromal cells produce a novel B cell stimulatory factor (BSF-TC) that induces proliferation of activated B cells.     

Identification of negative regulator of interleukin-3 (NIL-3) in bone marrow

We have reported that an inhibitor of interleukin-3 (NIL-3) is produced from murine bone marrow cells in response to excess stimulation of interleukin-3. In this report, we attempted the purification of the NIL-3 activity from bone marrow culture supernatant in the presence of interleukin-3. The purified NIL-3 activity was a protein with relative molecular weight of 54.5 kDa (SDS-PAGE), which inhibited the growth of IL-3 dependent DA-1 cell growth in a dose dependent manner. The N-terminal amino acid sequence of purified NIL-3 activity was determined to be homologous to beta-2 glycoprotein I (apolipoprotein H: APO-H). The gene expression of APO-H was detected by nested-PCR in STIL-3 C5-CM stimulated total bone marrow cells and STIL-3 C5-CM stimulated bone marrow fraction 2 (Fr. 2) which has been reported as a hematopoietic stem cell rich fraction. These observations indicate the possibility that the APO-H is the NIL-3 which was produced from bone marrow cells in response to excess IL-3 stimuli.     

Interleukin-6 production by the blast cells of acute myeloblastic leukemia: regulation by endogenous interleukin-1 and biological implications

Coordinate production of interleukin-1 beta (IL-1 beta) and granulocyte macrophage-colony stimulating factor (GM-CSF) or IL-6 by the blast cells of acute myeloblastic leukemia (AML) and normal peripheral blood leukocytes have been previously reported (van der Shoot et al.: Blood 74:2081-2087, 1989; Bradbury et al.: Leukemia 4:44-47 1990a, British Journal of Haematology 16:(in press), 1990b; Rodriguez-Cimadevilla et al.: Blood 76:1481-1489, 1990; Schindler et al.: Blood 75:40-47, 1990). In the present study, we show that IL-6 production by AML blasts is up-regulated by endogenously produced IL-1 beta. Neutralization of the endogenous source of IL-1 results in a significant decrease in IL-6 production, as determined by ELISA. Conversely, exposure of AML blasts to IL-1 alpha results in a significant increase in IL-6 production in 10 of 16 patient samples. Antibodies against IL-1 alpha and -beta also cause a drastic decrease in IL-6 and GM-CSF gene expression by the cells, suggesting that cytokine gene expression in AML blasts is driven, at least in part, by endogenous IL-1. The biologic significance of IL-6 production in culture of AML blasts has been addressed using a neutralizing antibody against IL-6. Our data indicate that IL-6 is important for the survival of clonogenic blasts in culture. In contrast, the survival of the total population of blasts is IL-6-independent, as assessed by the integrity of cellular DNA, even in the presence of anti-IL-6. These observations are consistent with the view that AML blasts might be organized as a lineage, with comparable hierarchy as in normal hemopoiesis and, perhaps, increased heterogeneity despite a homogenous appearance (McCulloch and Till: Blood Cells 7:63-77, 1981; Buick and McCulloch: Control of Animal Cell Proliferation. Academic Press, New York, vol. 1, pp. 25-57, 1985). Buick and McCulloch have identified a subpopulation of AML clonogenic cells with stem-cell-like properties, and suggested that the majority of blasts may have undergone a determination-like step. Our data indicate a marked difference in IL-6 requirement for cell survival between precursors and the majority of blasts, suggesting that IL-6 responsiveness may decrease following a determination-like event, i.e., the reduction in proliferative capacity.     

Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells

Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571-treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation.     

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.     

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.