Multifaceted mechanisms for cell survival and drug targeting in chronic myelogenous leukemia.

Treatment outcomes for chronic myelogenous leukemia (CML) have shown major improvements as a result of the development of the tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib for the disease-specific molecular target BCR-ABL1 tyrosine kinase (TK), but a cure of CML by BCR-ABL1 TKIs has been rarely achieved. CML cells are protected from cytotoxic insults, including those by TKIs, through various collaborative BCR-ABL1- mediated and -independent mechanisms, as well as cell-intrinsic and -extrinsic molecular mechanisms. These protective mechanisms include overlapping cell signaling pathways for normal hematopoietic proliferation, modulation of molecules associated with the BCL2 family protein-regulated programmed cell death pathway, autophagic cell protection capability, bone marrow environment-mediated cell protective signaling, abnormally upregulated genetic instability and other BCR-ABL1- independent kinase activities. To develop a more effective treatment strategy for a cure by means of total leukemic cell killing, a thorough understanding of how CML cells survive and resist cytotoxic insults is essential. In this article, we review current knowledge about multifaceted BCR-ABL1-related and -unrelated mechanisms for survival and death of CML cells and present suggestions for the development of new therapeutic strategies for complete elimination of residual CML cells during TKI treatment.

Cytochrome c induces caspase-dependent apoptosis in intact hematopoietic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling, MAP-kinase-kinase, and malignant change.

It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)-dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3-dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control.

Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells.

To determine the potential role of autocrine growth factor production in regulating primitive human hematopoietic cell development, we examined highly purified CD34+, c-Kit+ marrow mononuclear cells for expression of c-Kit ligand (KL) and stem cell tyrosine kinase 1 (stk1) ligand (STK1-L). Normal marrow mononuclear cells coexpressing CD34 and c-Kit were isolated by a combination of immunomagnetic bead isolation and fluorescence-activated cell sorting. Purified cells were then screened for expression of KL and stk1-L mRNA using a sensitive reverse transcription-polymerase chain reaction method. Using this approach, expression of both cytokine genes at the mRNA level was found in this highly enriched cell population. We then examined the functional significance of these mRNAs by inhibiting their expression with antisense (AS) oligodeoxynucleotides (ODN). In comparison to untreated or control ODN treated cells, inhibition of KL led to a 70% and 89% inhibition in burst-forming unit-erythroid (BFU-E) and colony-forming unit-Mix (CFU-Mix) colonies but had no significant effect on CFU-granulocyte-macrophage (CFU-GM) cloning efficiency. In contrast, inhibition of STK1-L alone had no effect on colony formation. However, when STK1-L AS ODN was combined with KL AS ODN, additive inhibition of CFU-GM and CFU-MIX but not of BFU-E colonies was observed. These findings, along with those of our previous studies showing inhibition of primitive hematopoietic cell growth with antisense ODN directed towards the stk1 receptor, suggest the possibility that both receptor/ligand axes regulate primitive hematopoietic cell growth via an autocrine growth loop.

Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro.

The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three-dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3-2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units-erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3-2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3-2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ.

Inhibition of stem cell factor-induced proliferation of primitive murine hematopoietic progenitor cells signaled through the 75-kilodalton tumor necrosis factor receptor. Regulation of c-kit and p53 expression.

TNF-alpha is a pleiotropic cytokine with stimulatory as well as inhibitory effects on hematopoiesis. We have previously demonstrated that TNF-alpha directly inhibits CSF-induced proliferation of primitive murine lineage-negative bone marrow progenitors (Lin-) and stem cell antigen-1 hematopoietic progenitors through the 75-kDa TNF receptor (TNF-R2), whereas TNF-alpha-induced inhibition of more committed Lin- progenitors is mediated through the 55-kDa TNF-R (TNF-R1), indicating a differential role of the two TNF-Rs in hematopoiesis. Numerous studies have demonstrated the ability of stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, to synergize with other hematopoietic growth factors, but little is known about cytokines capable of inhibiting hematopoiesis induced by SCF. While TNF-alpha has been demonstrated to enhance SCF-induced proliferation of myeloid leukemia blasts, the present report demonstrates that TNF-alpha, by signaling through TNF-R2, inhibits SCF-induced proliferation of normal murine Lin- and stem cell antigen-1 hematopoietic progenitors. SCF-stimulated proliferation of the hematopoietic cell line FDC-P1 was also potently inhibited by TNF-alpha and was accompanied by down-regulation of c-kit cell surface expression as well as c-kit mRNA levels. Finally, treatment of the FDC-P1 cell line with TNF-alpha resulted in increased levels of the tumor suppressor p53 mRNA, suggesting another mechanism by which hematopoietic effects of TNF-alpha may be mediated.

Regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist.

We investigated the effects of interferon-alpha (IFN-alpha) on the expression of cytokines by human bone marrow stromal cells. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and interleukin-1 beta (IL-1 beta) in stromal cell layers was induced by incubation with IL-1 alpha, tumor necrosis factor (TNF), or lipopolysaccharide (LPS). Addition of IFN-alpha to such stimulated cultures resulted in a strong downregulation of mRNA expression of GM-CSF and IL-1 beta. Similarly, the protein levels of GM-CSF and IL-1 beta were significantly reduced by IFN-alpha, whereas G-CSF production was only moderately inhibited. In contrast, IFN-alpha markedly stimulated the production of IL-1 receptor antagonist (IL-1RA) by stromal cells. The inhibition of cytokine expression resulted in a reduced hematopoietic activity of stromal cells, indicated by a reduced proliferation of the factor dependent cell line MO7e on IFN-alpha-treated stromal cells. In the presence of cycloheximide (CHX), IFN-alpha failed to inhibit IL-1 mRNA expression, whereas the regulation of GM-CSF and IL-1RA by IFN-alpha was not affected. Our results indicate that the myelosuppressive effects of IFN-alpha, as observed in therapeutic applications or associated with viral infections, are, in part, indirectly mediated by inhibition of the paracrine production of hematopoietic growth factors.

Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells.

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units-granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.

Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4.

In murines, interleukins (IL) 3, 4, 9, and 10, nerve growth factor, and stem cell factor induce or promote growth and differentiation of mast cells (MC). Increased stimulation and synergy was observed when combinations of cytokines were used. In man, no growth factor for human MCs had been identified until recently, when SCF was found to induce in vitro growth and differentiation of human MCs. In the present study, the effects of recombinant human IL-3 and IL-4 on SCF-dependent differentiation of human MCs from their circulating progenitor cells in long-term culture were analyzed. Surprisingly, both IL-3 and IL-4 were found to inhibit SCF-dependent formation of human MCs (SCF, 100 ng/ml: 36.4 +/- 18.7 x 10(3)/ml; SCF + IL-3, 100 U/ml: 23.4 +/- 4.2 x 10(3)/ml; SCF + IL-4, 100 U/ml: 7.4 +/- 4.4 x 10(3)/ml) and synthesis of MC tryptase (SCF, 100 ng/ml: 73.2 +/- 17.6 ng/ml; SCF + IL-3, 100 U/ml: 10.8 +/- 3.1 ng/ml [p < 0.01]; SCF + IL-4, 100 U/ml: 8.1 +/- 1.5 ng/ml, [p = 0.02]). The inhibitory effects of these cytokines on SCF-dependent formation of human MCs were associated with an increase in the number of macrophages (IL-3) or lymphocytes (IL-4) in the same cultures and may be due to competitive recruitment of cells from a pool of multilineage hematopoietic progenitor cells.

Transforming growth factor-beta prevents stem cell factor-mediated rescue of mast cells from apoptosis after IL-3 deprivation.

IL-3-dependent mast cells undergo apoptosis upon removal of IL-3, an event that is prevented by the addition of stem cell factor (SCF) acting through its receptor c-kit, suggesting that SCF provides a mechanism to allow mast cells to survive and to differentiate in tissues in the relative absence of IL-3. This observation is consistent with the thesis that the microenvironment, in part, controls mast cell number and viability by modulating SCF production and release. The purpose of the present study was to determine whether a second factor, TGF-beta 1, was capable of modifying the SCF-mediated survival pathway. TGF-beta 1 (1 and 10 ng/ml), known to be an important regulator of cell growth and function, did inhibit the SCF-mediated rescue from apoptosis in IL-3-deprived mast cells. TGF-beta 1 exerted its inhibitory effect on SCF-mediated rescue from apoptosis, even when added 4 h after the addition of SCF. In contrast, TGF-beta 1 had no substantial effect on the viability of mast cells that were grown in the presence of IL-3. TGF-beta 1 also had no noticeable effect on viability and proliferation of a growth factor-independent mast cell line. The inhibitory effect of TGF-beta 1 was neutralized by specific anti-TGF-beta mAb. TGF-beta 1 did not affect the expression of c-kit, as determined by using flow cytometric analysis of mast cells labeled with FITC-conjugated anti-c-kit. These results demonstrate how SCF and TGF-beta may act in concert to regulate mast cell numbers under physiologic or pathologic conditions.

Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors.

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-alpha has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-alpha and SCF. Here, we demonstrate that TNF-alpha potently and directly inhibits SCF-stimulated proliferation of CD34+ hematopoietic progenitor cells. Furthermore, TNF-alpha blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-alpha, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNF-alpha were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-alpha downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well.

Protein kinase C-dependent release of a functional whole extracellular domain of the mast cell growth factor (MGF) receptor by MGF-dependent human myeloid cells.

The mast cell growth factor (MGF) affects migration, proliferation and differentiation of erythroid and myeloid progenitor cells by binding to a transmembrane receptor tyrosine kinase encoded by the c-Kit proto-oncogene. By using MGF-dependent human myeloid cell lines (M-07e and TF-1), here we show that a Kit-related 100 kDa protein is associated with the cell but it undergoes release into the medium upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. Immunological analysis with a series of antibodies to Kit indicated that the released protein (p100Kit) contains the whole glycosylated extracellular portion of the transmembrane Kit protein (p145Kit). The secreted protein retained the ability to specifically bind MGF. Moreover, p100Kit was able to block the mitogenic effect of MGF on cultured M-07e cells, suggesting that the soluble protein may function as a physiological antagonist of MGF.

Transforming growth factor beta abrogates the effects of hematopoietins on eosinophils and induces their apoptosis.

Hematopoietins, interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) have previously been shown to prolong eosinophil survival and abrogate apoptosis. The objective of this study was to investigate the effect of transforming growth factor beta (TGF-beta) on eosinophil survival and apoptosis. Eosinophils from peripheral blood of mildly eosinophilic donors were isolated to > 97% purity using discontinuous Percoll density gradient. Eosinophils were cultured with hematopoietins with or without TGF-beta for 4 d and their viability was assessed. We confirmed previous observations that hematopoietins prolonged eosinophil survival and inhibited apoptosis. TGF-beta at concentrations > or = 10(-12) M abrogated the survival-prolonging effects of hematopoietins in a dose-dependent manner and induced apoptosis as determined by DNA fragmentation in agarose gels. The effect of TGF-beta was blocked by an anti-TGF-beta antibody. The anti-TGF-beta antibody also prolonged eosinophil survival on its own. The culture of eosinophils with IL-3 and GM-CSF stimulated the synthesis of GM-CSF and IL-5, respectively, suggesting an autocrine mechanism of growth factor production. TGF-beta inhibited the synthesis of GM-CSF and IL-5 by eosinophils. TGF-beta did not have any effect on the expression of GM-CSF receptors on eosinophils. We also studied the effect of TGF-beta on eosinophil function and found that TGF-beta inhibited the release of eosinophil peroxidase. Thus, TGF-beta seems to inhibit eosinophil survival and function. The inhibition of endogenous synthesis of hematopoietins may be one mechanism by which TGF-beta blocks eosinophil survival and induces apoptosis.

Autocrine transforming growth factor beta 1 blocks colony formation and progenitor cell generation by hemopoietic stem cells stimulated with steel factor.

The ability of Steel Factor (SF) to stimulate colony formation and progenitor cell generation by hemopoietic stem cells (HSCs) in vitro in the absence of interleukin 3 (IL-3) was investigated. IL-3 was required for HSC proliferation, and no or restricted proliferation occurred in the presence of SF, IL-6, IL-11, or IL-12 as single factors or in combination. Neutralizing concentrations of anti-transforming growth factor (TGF)-beta 1 antibodies enhanced progenitor cell generation 2-3-fold in the presence of IL-3, but 75 to over 300-fold when cultures contained at least SF in the absence of IL-3. Exogenous TGF-beta 1 fully abrogated the antibody effects. In the presence of antibodies to TGF-beta 1, SF alone stimulated the delayed formation of small blast cell colonies and SF synergized with IL-6, IL-11, or IL-12 to greatly hasten colony formation, enhance colony number and size, and increase colony forming unit-culture (CFU-C) output from suspension cultures of enriched HSC populations. Secondary CFU-C colonies were significantly larger when IL-3 was absent during the suspension culture phase. Single cell and limiting dilution analysis using a homogenous colony forming unit-spleen (CFU-S) day-12 population and an 800-fold enriched long-term repopulating HSC fraction, respectively, indicated that TGF-beta 1 was an autocrine product of these HSC subsets. Addition of nucleosides, insulin, extra glucose, or serum could not replace the effects of the anti-TGF-beta 1 antibody. While these data offer one possible explanation for reports on the inability of SF to stimulate HSC proliferation, they present the basis for a novel model of the regulation of HSC activation wherein: 1) close-range interactions of HSCs with mesenchymal stromal cells do not exclusively determine maintenance of HSC quiescence; 2) competence acquisition by dormant HSCs may involve the down-regulation or inactivation of autocrine TGF-beta 1; and 3) SF may act as a primary growth factor rather than exclusively as a synergistic cytokine.

Hematopoietic growth factors: current knowledge, future prospects.

The introduction of hematopoietic growth factors into clinical medicine represents one of the more exciting developments in oncology in the past several years. The identification, gene cloning, and large-scale production of hematopoietic growth factors represent important examples of the practical benefits that may accrue from application of the sophisticated technology derived from recombinant DNA research. Research, both at the bench and by the bedside, has proceeded at an extraordinarily rapid pace in this field over the past five years, leading to an abundance of new information, novel promising agents, and important clinical controversies related to the biology and appropriate clinical applications of hematopoietic growth factors. With these agents, for the first time in history, the production of human blood cells can be systematically manipulated in vivo in an effort to optimize physiology beyond the endogenous host response. Additionally, investigators utilizing purified hematopoietic growth factors as reagents may provide crucial insights into the mechanisms of blood cell production in health and in various disease states. This review aims to summarize the current state of knowledge regarding the control of blood cell production by specific factors and to put these data in the context of clinical medicine. The emphasis will be on factors that primarily influence myeloid (rather than lymphoid) cell growth, differentiation, and activation, and the clinical focus will be on applications in oncologic therapeutics and in the treatment of primary hematologic disorders. By reviewing what we know and what has already been done, we may be better able to define the important questions that remain and to formulate the means to answer our current uncertainties about the activities and clinical uses of hematopoietic growth factors.

A recombinant ectodomain of the receptor for the stem cell factor (SCF) retains ligand-induced receptor dimerization and antagonizes SCF-stimulated cellular responses.

The stem cell factor (SCF) is a polypeptide ligand that is essential for the development of germ cells, hematopoietic progenitor cells, and melanocyte precursors. It binds to a tyrosine kinase membrane receptor that is encoded by the c-kit proto-oncogene. We have constructed an expression vector that directs the synthesis of the entire extracellular ligand-binding domain of the Kit/SCF receptor. When expressed and amplified in Chinese hamster ovary cells, a secreted 90-kDa glycoprotein could be harvested from the growth medium of the cells in a soluble form. This extracellular portion of the Kit/SCF receptor, denoted Kit-X, was recognized by antibodies specific to the SCF receptor; and when injected into animals, it raised antibodies that were reactive with the complete membrane form of the receptor. Direct binding and covalent cross-linking of radiolabeled SCF showed that Kit-X fully retained high affinity ligand binding and also underwent efficient dimerization in the presence of the ligand. The capacity of Kit-X to act as an antagonist of SCF was assayed on cultured cells that overexpress the receptor. Simultaneous addition of SCF and Kit-X to these cells resulted in a stoichiometric inhibition of SCF binding and a consequent decrease in autophosphorylation of the SCF receptor on tyrosine residues. The inhibition extended to later SCF-mediated responses, including the association of the receptor with phosphatidylinositol 3'-kinase and coupling to the Raf1 protein kinase. These results indicate that the recombinant ectodomain of the Kit-SCF receptor can be used as a specific antagonist of SCF actions and may enable detailed molecular analysis of ligand-receptor interactions.

Transforming growth factor beta inhibits the action of stem cell factor on mouse and human hematopoietic progenitors.

In agar culture of post 5-fluorouracil mouse bone marrow cells (FUBM), recombinant rat stem cell factor (rrSCF) synergizes with granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) or interleukin-6 (IL-6) to stimulate primitive progenitor cells (HPP-CFCs). The addition of recombinant human transforming growth factor beta (rhTGF-beta) to cultures of FUBM containing rrSCF plus rhG-CSF, rrSCF plus recombinant murine (rm)IL-3, or rrSCF plus rhIL-6 resulted in 100% inhibition of colony formation. Highly enriched populations of primitive bone marrow cells were obtained by isolating lineage negative (Lin-), Sca-1-positive (Sca-1+) cells from normal mouse bone marrow. RhTGF-beta inhibited 90% of colony formation stimulated by rrSCF plus rmIL-3 in agar culture of the Sca-1+ cells. RhTGF-beta also inhibited colony formation in agar culture of post FU human bone marrow cells. The synergistic increase in colony formation obtained with recombinant human SCF (rhSCF) plus rhGM-CSF and rhSCF plus rhIL-3 was inhibited by rhTGF-beta (approx. 60% and 87% inhibition, respectively). RhTGF-beta also totally inhibited the erythroid colony formation stimulated by rhSCF plus recombinant human erythropoietin (rhEpo). These data demonstrate that TGF-beta inhibits SCF-stimulated colony formation of mouse and human BM. This inhibition on progenitor cells appears to be a direct action of TGF-beta and is consistent with the target cells of SCF being more primitive progenitors than the CFCs stimulated by the CSFs alone.