Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colony-stimulating factor.

The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 micrograms/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32 +/- 7 to 79 +/- 16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43 +/- 20 to 82 +/- 11%) and day 7 CFU-GM (from 41 +/- 14 to 56 +/- 20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26 +/- 14 to 41 +/- 6%, and that of erythroblasts increased from 25 +/- 12 to 30 +/- 12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents.

Insensitivity of chronic myeloid leukemia cells to inhibition of growth by prostaglandin E1.

The influence of E prostaglandins on the in vitro growth of chronic myeloid leukemia (CML)-committed granulopoietic precursors [colony-forming unit-culture (CFU-C)] has been investigated in a double-layer agar system in which CFU-C growth was stimulated by adherent monocytes. Addition of the prostaglandin synthesis inhibitor indomethacin to the feeder layer significantly increased the number of normal CFU-C, whereas CML CFU-C were unaffected. Exogenous prostaglandin E1 inhibited CML CFU-C growth at concentrations 1000-fold higher than those necessary to produce a similar effect on normal CFU-C. These data point to a lower than normal sensitivity of CML-committed granulopoietic precursors. It is suggested that derangement of the responsiveness of CML cells to prostaglandin regulation may play a role in the pathogenesis of uncontrolled leukemic proliferation.

Opposite effect of tumor necrosis factor alpha on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors.

The effect of recombinant human tumor necrosis factor alpha (TNF-alpha) on normal and chronic myeloid leukemia granulocyte-macrophage progenitors (CFU-GM) growing in semisolid agar cultures in the presence of recombinant granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor was studied. Granulocyte-macrophage colony-stimulating factor-dependent growth of normal and chronic myeloid leukemia bone marrow CFU-GM was greatly enhanced by TNF-alpha at doses of 0.1 to 100 units/ml. Growth enhancement included neutrophil, eosinophil, and monocyte-macrophage colonies and clusters at 7 and 14 days of culture. Since similar results were achieved with highly enriched progenitor cell populations, devoid of accessory cells, an indirect effect on CFU-GM growth through the release by accessory cells of other cytokines upon TNF-alpha stimulation was thus ruled out. By contrast, the same doses of TNF-alpha inhibited the growth of normal CFU-GM in granulocyte colony-stimulating factor-dependent cultures. Taken together, our findings indicate that the final effect of TNF-alpha on normal bone marrow granulocyte-macrophage progenitor growth is dependent on the specific growth factor interacting with it, and that both normal and chronic myeloid leukemia CFU-GM are equally responsive to the combined effects of TNF-alpha and a given colony-stimulating factor.

Expression of HLA class II (DR, DQ) determinants by normal and chronic myeloid leukemia granulocyte/monocyte progenitors.

It has been suggested that the expression of certain HLA class II antigens stemming from three distinct loci (DR, DP, and DQ) is important not only in the regulation of the immune response but also on the response of hemopoietic precursors to factors inhibiting myelopoiesis. Changes in the expression of DR antigens may be involved in the pathogenesis of altered cell proliferation in chronic myeloid leukemia, since they result in decreased sensitivity of the colony forming units, granulocyte-macrophage to prostaglandin E and acidic isoferritins. In studies using monoclonal antibodies against monomorphic DR or DQ determinants, in a complement-dependent cytotoxic assay, it was found that nearly all normal and chronic myeloid leukemia bone marrow colony forming units, granulocyte-macrophage express DR antigens. The dose response curve was similar for both normal and leukemic precursors. Leukemic peripheral blood precursors were more sensitive than were normal peripheral blood precursors. Normal colony forming units, granulocyte-macrophage did not express DQ antigens, whereas these were expressed in varying quantities by leukemic cells. This study shows that, in the patients we studied, leukemic cells express DR antigens in amounts comparable to normal. In addition, varying amounts of DQ antigens may be observed on leukemic but not on normal progenitors, perhaps as a consequence of an increase in the number of antigens also expressed by normal cells, though in an amount below the detection threshold of cytotoxicity techniques.

Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts.

The stem cell tyrosine kinase 1 (STK1) protein is the human homologue of the murine FLT3 gene product, a receptor belonging to the FMS/KIT family. FLT3 and KIT with their ligands control the growth and differentiation of early human hemopoietic cells. In the present study, 16 cases of acute myeloid leukemia (AML) were examined by flow cytometry for cell surface expression of FLT3 and KIT receptors. All cases were also tested for their proliferative response to human FLT3 ligand (FL) and KIT ligand (KL) and for colony formation in the presence of single or associated cytokines. Among 16 AML cases tested, 10/16 expressed FLT3 receptor and 12/16 expressed KIT receptor, without any correlation with FAB subtype. FL and KL stimulated the proliferation of leukemic blasts in 11/16 AML cases (including five FLT3 or KIT receptor-negative cases), with an additive effect when added simultaneously. By contrast, some receptor-expressing AMLs did not display significant proliferative responses to their respective ligands. FL and KL as single factors induced or significantly increased the colony formation by clonogenic precursor cells respectively in eight and six of 13 cases tested. In some cases growth factor association significantly enhanced colony growth. Taken together these observations provide evidence that the pattern of FLT3 and KIT receptor expression is extremely variable among the AMLs and that receptor presence is not necessarily combined with proliferative and clonogenic response or vice versa.

Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.

Cord blood (CB) is an attractive alternative to bone marrow or peripheral blood as a source of transplantable hematopoietic tissue. However, because of the reduced volume, the stem cell content is limited; therefore its use as a graft for adult patients might require ex vivo manipulations. Two systems have been described that identify these stem cell populations in vitro in both mice and humans: (1) the long-term culture-initiating cells (LTC-IC), thus named because of their ability to support the growth of hematopoietic colonies (colony-forming cell (CFC)) for 5-6 weeks when co-cultured on stromal layers; (2) the generation of hematopoietic progenitors (CFC) from stroma-free liquid cultures for extended periods of time, which provides further indirect evidence of the presence of primitive stem cells. Both systems detect largely overlapping but not identical populations of stem cells. Thus the identification of the growth factor requirements for the maintenance and amplification of both systems is relevant. On this basis, analysis of the effects of 18 cytokine combinations on stroma-free liquid cultures of CB CD34+ cells, showed that: (1) after 7- and 14 day-incubation periods, several growth factor combinations expanded the LTC-IC pool to a similar extent; as compared to the LTC-IC, the generation of CFC was not impressive; (2) time-course analysis of the LTC-IC expansion demonstrated that, by extending the incubation period, only a few growth factor combinations, containing FL, TPO, KL and IL6, could support a further, increasingly greater LTC-IC expansion (up to 270000-fold of the initial value). In similar culture conditions, CFC production underwent continuous expansion, which persisted for over 7 months and reached values of one million-fold of the initial value. The simultaneous presence of FL and TPO was both necessary and sufficient to support this phenomenon. The addition of KL+/-IL6 did not appear to substantially modify the extent of LTC-IC expansion; nevertheless, it played an important role in sustaining an even more massive and prolonged output of CFU-GM, CFU-Mk and BFU/CFU-GEMM (up to 100 million-fold); (3) the presence of IL3 was found to be negative, in that it inhibited both the extent of LTC-IC expansion and the long-term generation of CFC. Thus, FL and TPO appear as two unique growth factors that preferentially support the self-renewal of primitive stem cells; the additional presence of KL and IL6 seems to enhance the proliferative potential of at least one subpopulation of daughter stem cells, which may follow three differentiation pathways. Far from being definitive, our data demonstrated that massive stem cell expansion, in cord blood, can be obtained in reasonably well-defined culture conditions. This could represent an initial step towards larger scale cultures for transplantation and gene therapy protocols.

Stem cell factor improvement of proliferation and maintenance of hemopoietic progenitors in myelodysplastic syndromes.

Human recombinant stem cell factor (rSCF) was tested for its capability of improving the defective growth of hemopoietic progenitors in 28 cases of myelodysplastic syndromes (MDS). In vitro growth and response to rSCF were quite variable. However, in most cases, rSCF stimulated CFU-GM growth induced by rG-CSF, rGM-CSF, rIL-3, 5637 conditioned medium (50-1400% enhancement). rSCF effect was slightly more evident on day 14 CFU-GM and in the presence of rIL-3. BFU-E growth induced by rEPO or rIL-3 + rEPO was enhanced by rSCF in about 50% of cases, in linear correlation with the levels of patients' hemoglobin. rSCF did not increase CFU-E growth, whereas it slightly stimulated CFU-Mk in 33% of the cases. EPO, SCF and, particularly, their combination, enhanced the recovery of normal CFU-E and BFU-E after 7 days of liquid culture. This was less evident in cultures of MDS patients. Conversely, CFU-GM generation in long term liquid cultures, although highly variable, was stimulated by rSCF and, above all, by rSCF + rG-CSF, similarly to what was observed with normal bone marrow samples. SCF seems to enhance in vitro erythropoiesis only in MDS cases presenting without severe anemia. It has little effect on megakaryocytopoiesis, while it seems to be more active on CFU-GM growth and maintenance.

In vivo effect of human granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil GM-CSF receptors.

The effect of in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophils GM-CSF receptor, was investigated in patients with neoplastic diseases and normal hematopoiesis. Patients were divided into two groups. Group A received a single dose of rhGM-CSF (5 micrograms/kg/day) and receptor studies were performed 90 min and 48 h after treatment. Group B received three doses, administered subcutaneously every 24 h and receptor studies were performed 90 min after first injection and 24 h after the last. Before treatment neutrophils only displayed high-affinity receptors (KD 85 +/- 53 pM; number of receptors/cell 1318 +/- 567). The first injection of rhGM-CSF produced a transient leucopenia and the internalization of GM-CSF receptor on neutrophils in both groups of patients: 90 min after s.c. administration receptors could not be detected with conventional binding studies. In group A patients, 48 h after a single dose of rhGM-CSF, receptors, albeit with a decreased affinity (KD = 240 +/- 131 pM; number of receptors/cell 783 +/- 494) were again expressed. In group B patients, 24 h after the last rhGM-CSF injection, low intermediate affinity receptors not present before treatment appeared (KD 720 +/- 175 pM; number of receptor/cell 1222 +/- 179). They were associated with a low number of high affinity receptors (KD = 9 +/- 4 pM; number of receptors/cell 106 +/- 44). These observations indicate that more than one type of GM-CSF receptor may exist on neutrophils. It may be suggested that in vivo the regulation of the GM-CSF receptor is different from that in vitro and is related to the presence of the cytokine in patient blood.

In vivo effect of granulocyte-macrophage colony-stimulating factor on the kinetics of human acute myeloid leukemia cells.

Granulocyte-macrophage colony-stimulating factor, (GM-CSF) was given at 8 micrograms/kg daily by continuous i.v. infusion for 72 h to six patients with acute myeloid leukemia (AML) in expansion and one with chronic myeloid leukemia in blastic crisis to determine whether it was possible to augment the proliferative activity of the neoplastic population. The percentage of marrow blasts in S phase (labeling index, LI) was increased in five patients (1.3-, 1.5-, 1.9-, 2.3- and 3.2-fold change). The increase in LI was similar 24 and 48 h after beginning GM-CSF. The RNA Index also increased in patients who showed an increased LI, suggesting that GM-CSF had recruited quiescent neoplastic cells into the cell cycle. Forty eight hours after beginning GM-CSF, chemotherapy was started. The fate of S phase cells, labeled in vivo with bromodeoxyuridine (BrdU) immediately before cytostatic treatment, was monitored. BrdU positive cells were identified by fluorescent antibody for up to 28 days. A preferential killing of BrdU (S phase) cells was observed in 5/7 patients who obtained a complete remission, whereas this was not apparent in the two patients who achieved only a partial remission. Chemotherapy induced a rapid and profound aplasia; its duration, however, was not significantly different from that observed in historical controls. GM-CSF may have a potential role in the treatment of AML, as this study shows that it recruits leukemic cells into the cell cycle without adversely prolonging aplasia after cycle-specific therapy.

Megakaryocyte growth and development factor (MGDF)-induced acute leukemia cell proliferation and clonal growth is associated with functional c-mpl.

The effects of human recombinant megakaryocyte growth and development factor (MGDF) (also known as thrombopoietin (TPO)), alone or in combination with other growth factors, on the proliferation and on the clonal growth of clonogenic progenitors from 24 acute myeloblastic leukemia (AML) patients were evaluated. A significant proliferative response to MGDF alone (proliferation index > 1.5) was observed in nine of 23 cases; the responding cases belonged to all FAB subtypes. However, the greatest response (proliferation index > 7) was found in one M6 and in one M7 case. MGDF also enhanced interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), c-kit ligand (KL) and FLT3 ligand (FL) stimulated blast cell proliferation. MGDF as a single factor induced or significantly enhanced colony formation by clonogenic precursor cells in 12 of 14 AML cases. MGDF strongly increased KL-induced leukemic colony growth in seven cases, whereas it only moderately enhanced IL-3- or GM-CSF-induced colony growth. The analysis of tyrosine phosphorylated protein(s) upon MGDF stimulation in fresh AML cells was also performed. The results demonstrated a band of approximately 90 kDa phosphorylated protein(s) upon MGDF stimulation in AML responsive cases, but not in unresponsive ones. Taken together the present findings suggest that, in a consistent proportion of AML cases, MGDF stimulates blast cell growth and induces tyrosine protein phosphorylation.

Detection of basophils growing in semisolid agar culture.

The production of basophils in semisolid agar cultures form normal and chronic myeloid leukemia (CML) committed granulocyte-macrophage precursors was investigated using an original whole-dish staining technique with toluidine blue which produces a specific metachromasia in basophils. As additional proof of the basophilic nature of metachromatic cells, their degranulation after challenge with C5a anaphylotoxin and Synachten was observed. Our studies show that few basophils are produced in cultures form normal bone marrow. CML CFUc produce more basophils, their number being roughly correlated with the degree of basophilia. We observed only clusters composed by a pure basophilic population, while larger aggregates in which basophils could be detected were composed also by other granulocytic cells.

An in vitro study of basophil production in chronic myeloproliferative disorders.

An examination was carried out of certain parameters of basophil production in semisolid agar cultures by granulomonocyte precursors (CFU-C) from normal subjects and from patients with chronic myeloproliferative disorders. Basophils were rare in the first four days of culture, after which their number increased gradually to reach a plateau on about the 12th day. Pure basophil colonies were frequent only in cultures from a patient with marked basophilia. In the other cases the colonies also included other cells of the granulomonocyte pathway. There was a linear correlation between basophilia in vivo and basophil production in vitro. The latter, however, was influenced by the type of colony-stimulating activity (CSA) used. Different CSA sources, inducing the production of a comparable number of colonies, did not stimulate basophilic differentiation in the same way. Serum from patients with chronic myelocytic leukemia and varying degrees of basophilia did not have a significant effect on autologous in vitro basophilopoiesis, nor did it increase that of normal CFU-C.

Prostaglandin E counteracts the gamma interferon induction of major histocompatibility complex class-II antigens on U937 cells and induction of responsiveness of U937 colony-forming cells to suppression by lactoferrin, transferrin, acidic isoferritins, and prostaglandin E.

The established human monoblast or early monocyte cell line, U937, was evaluated for modulating influences of prostaglandin E2 (PGE2) on human gamma interferon (HuIFN gamma) induction of MHC class-II (Ia) antigens on U937 cells and the HuIFN gamma induction of responsiveness of U937 colony-forming cells (CFC) to inhibition by lactoferrin (LF), transferrin (TF), acidic isoferritins (AIF), and prostaglandin E (PGE). U937 CFC were induced to a state of responsiveness to the suppressive influences of PGE by HuIFN gamma. When MHC class-II antigens were induced on U937 cells and the cells sorted on the fluorescence activated cell sorter (FACS) IV into positive and negative cells, colony formation by the MHC class-II antigen+ population of cells was suppressed by LF, TF, AIF, and PGE2. Colony formation by the sorted population of MHC class-II antigen- cells was not influenced significantly by LF, TF, AIF, or PGE2. When PGE was present in the suspension culture for 72 h with U937 cells exposed to HuIFN gamma plus indomethacin, it blocked the induction of MHC class-II antigens as well as the associated inhibition of U937 CFC by LF, TF, AIF, and PGE2.

In vitro reappearance of myeloid progenitors killed by mafosfamide.

Cyclophosphamide derivatives active in vitro, such as mafosfamide, are potentially capable of reducing the number of leukemic cells remaining in marrow explanted for autografting. Although this treatment kills nearly all the committed hemopoietic progenitors, it does not prevent the reinstatement of hemopoiesis after chemoradiotherapy. This points to the persistence of more immature hemopoietic progenitors not detectable with current semi-solid culture techniques and resistant to cytotoxic treatment. Treatment of normal marrow with 80-140 micrograms/ml mafosfamide is followed in medium-term cultures by a gradual and dose-dependent reduction in total cellularity, whereas granulomonocyte progenitors (CFU-GM), virtually absent at the start of the culture, progressively reappear. The quantity of progenitors present after day 14 in liquid culture is, however, less in the treated marrows than in the controls, and the reappearance of CFU-GM is inversely related to the mafosfamide dose. In addition, the recovery of the more immature (day-14) CFU-GM is greater than that of the more mature (day 7) CFU-GM.

Purified human transferrin and "transferrin" released from sorted T8+ lymphocytes suppress release of granulocyte-macrophage colony-stimulating factors from sorted T4+ lymphocytes stimulated by phytohemagglutinin.

Nonadherent, low-density E-rosette-positive human peripheral blood cells were separated into T4+ and T8+ lymphocytes by immuno-fluorescence-activated cell sorting (FACS) with monoclonal antibodies OKT4 and OKT8. Both T4+ and T8+ lymphocytes released granulocyte-macrophage colony-stimulating factors (GM-CSF) in response to phytohemagglutinin (PHA). Purified iron-saturated human transferrin (TF) suppressed release of GM-CSF only from the T4+ subset of lymphocytes. A TF-type inhibitory activity was released from the T8+ subset of lymphocytes alone, and this inhibitory activity, as well as that in purified TF, was inactivated by preincubation with antihuman TF monoclonal antibody (HT/1). These studies suggest that, at least in vitro, subsets of T-lymphocytes and TF may be involved in the regulation of myelopoiesis.

Gamma interferon induces colony-forming cells of the human monoblast cell line U937 to respond to inhibition by lactoferrin, transferrin, and acidic isoferritins.

Human gamma interferon (HuIFN gamma) was assessed for its capacities to induce MHC class-II antigens on U937 cells and to induce responsiveness of U937 colony-forming cells (CFC) to the suppressive influences of lactoferrin (LF), transferrin (TF), and acidic isoferritins (AIF). U937 cells grown in suspension culture for many years demonstrated variable percentages of MHC class-II antigen+ cells (6%-42%) as determined by analysis with monoclonal anti-MHC class-II and the FACS IV when checked at different times. The percentage of U937 cells positive for MHC class-II antigens, as well as the density distribution of MHC class-II antigens on these cells, was increased by preincubating the cells for 72 h in the presence of 10(-6) M indomethacin and increasing concentrations of natural HuIFN gamma up to 20-40 U/ml. Colony formation by cells preincubated in control medium plus indomethacin for 72 h was not decreased by treating cells with monoclonal anti-MHC class-II plus complement (C'), high specific activity tritiated thymidine (3HTdr), LF, TF, or AIF. After preincubation of U937 cells with natural HuIFN gamma plus indomethacin in suspension culture for 72 h, colony formation in semisolid medium was reduced 40%-50% by treating the cells with anti-MHC class-II plus C', 3HTdr, LF, TF, or AIF. Colony formation was not reduced further by LF, TF, or AIF, after cells were pretreated with anti-MHC class-II (1:200 dilution) plus C' or 3HTdr. Increasing concentrations of HuIFN gamma up to 20 U/ml increased the percentage of MHC class-II antigen+ U937 CFC as well as the sensitivity of U937 CFC to suppression by LF, TF, and AIF. The inducing activities of natural HuIFN gamma were due to the IFN gamma itself since the inducing activity of natural HuIFN gamma was inactivated by pretreatment with a monoclonal antibody against natural HuIFN gamma. Also the inducing effects were mimicked by recombinant HuIFN gamma. The suppressive effects of LF, TF, and AIF on colony formation were blocked by treating the cells with monoclonal anti-MHC class-II (1:50 dilution, but not 1:200 dilution) in the absence of C'. The suppressive effect of TF only was blocked by pretreating cells with a monoclonal antibody against the TF receptor. U937 cells can be used as a model to study the regulatory mechanisms of action of HuIFN gamma, LF, TF, and AIF.

Responsiveness of highly enriched CFU-GM subpopulations from bone marrow, peripheral blood, and cord blood to hemopoietic growth inhibitors.

Human early and late granulocyte-monocyte progenitors (granulocyte-macrophage colony-forming units, CFU-GM), depleted of accessory cells, were physically separated using an antimyeloid monoclonal antibody (DS1.1). They were separately cultured at optimal growth conditions and tested for responsiveness to prostaglandin E2 (PGE2), recombinant tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta-1 (TGF beta 1). Late (DS1.1+) CFU-GM displayed the highest sensitivity to PGE2 and TNF alpha, the first significant inhibition being evident at 10(-9)M PGE2 and 1 U/ml TNF alpha. Conversely, their growth was stimulated (211%-217%) by 0.25-2.5 ng/ml TGF beta 1. Early (DS1.1-) marrow CFU-GM evidenced a lower sensitivity to PGE2 and TNF alpha. Their growth, however, was inhibited by 0.25-2.5 ng/ml TGF beta 1. Early CFU-GM constitute the totality of peripheral blood myeloid progenitors. Cord blood CFU-GM were also demonstrated here to be entirely DS1.1-. Both adult and cord blood CFU-GM displayed the highest resistance to PGE2 and TNF alpha. By contrast, they showed the maximum sensitivity to growth inhibition by TGF beta 1, active at 0.025-0.25 ng/ml. For the first time, therefore, highly purified subsets of human CFU-GM were separated that displayed a different responsiveness to well-defined growth-regulatory molecules. Our results indicate that TGF beta 1 has a dual activity; it is inhibitory on early and stimulatory on late CFU-GM, whereas PGE2 and TNF alpha preferentially inhibit late CFU-GM growth.

The involvement of human-nuc gene in polyploidization of K562 cell line.

During megakaryocyte differentiation, the immature megakaryocyte increases its ploidy to a 2(x) DNA content by a process called endomitosis. This leads to the formation of a giant cell, the mature megakaryocyte, which gives rise to platelets. We investigated the role of human-nuc (h-nuc), a gene involved in septum formation in karyokynesis in yeast, during megakaryocytic polyploidization. Nocodazole and 12-O-tetradecanoylphorbol-13-acetate (TPA) were used to induce megakaryocytic differentiation in K562 cell line. The ploidy distribution and CD41 expression of treated K562 cells were evaluated by flow cytometry. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we analyzed the h-nuc mRNA expression on treated K562 cells. Mature megakaryocyte-like polyploid cells were detected at day 5-7 of treatment with nocodazole. TPA also had a similar effect on K562 cells, but it was much weaker than that of nocodazole. The analysis of ploidy of nocodazole-treated K562 cells showed that nocodazole preferentially induced polyploidization of K562 cell line with a pronounced increase of the cells 8N at day 7 of culture. Expression of CD41, a differentiation-related phenotype, was significantly induced by TPA after 7 days of treatment, showing that functional maturation was mainly induced by TPA. In contrast, there was no significant increase in CD41 expression in nocodazole-treated K562 cells, suggesting that polyploidization and functional maturation are separately regulated during megakaryocytopoiesis. RT-PCR analysis indicated that h-nuc mRNA increased after 72 hours in the presence of nocodazole, preceding the induction of polyploidization. Our data indicate that h-nuc might play a role in polyploidization during megakaryocytic differentiation via inhibition of septum formation.

The effects of human FLT3 ligand on in vitro human megakaryocytopoiesis.

The human homolog of the murine flt3/flk2 gene product is a tyrosine kinase receptor that plays a role in regulating the proliferation and differentiation of cells in the hematopoietic system. Using a plasma-clot clonal assay and a long-term bone marrow culture (LTBMC) system, we studied the effects of the recently cloned human flt3 ligand (FL) alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or stem cell factor (c-kit ligand [KL]) on human megakaryocytopoiesis. The effects of FL on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. FL alone had no megakaryocytic colony-stimulating activity (MK-CSA), but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. FL synergized with IL-3 at the level of both CFU-MK and BFU-MK and with GM-CSF and KL at the level of CFU-MK. Although FL alone exhibited a limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3 and KL, alone or in association, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that FL plays a significant role in this process by amplifying the MK-CSA of GM-CSF, IL-3, and KL.

Prostaglandin E acts at two levels to enhance colony formation in vitro by erythroid (BFU-E) progenitor cells.

The prostaglandin E (PGE) enhancement of erythroid colony formation by human bone marrow erythroid progenitor cells (BFU-E) is mediated by a T8+ subset of lymphocytes. Medium was conditioned by bone marrow and blood T-lymphocytes and T-lymphocyte subsets (T8+, T8-, T4+, and T4- cells) in the absence or presence of PGE1 in order to determine if the cells could release a cell-free source of erythroid colony enhancing activity and what the conditions for this release would be. The T-lymphocyte conditioned medium was assayed for its effects on erythroid colony formation by nonadherent low-density T-lymphocyte depleted (NALT-) bone marrow cells plated in the presence of erythropoietin, hemin, phytohemagglutinin-stimulated leukocyte conditioned medium, or medium conditioned by 5637 cells, in the absence or presence of PGE1 and in the presence or absence of serum. PGE1 induced the release of an erythroid colony enhancing activity from the T8+ and T4-, but not from the T8- and T4+ subsets of lymphocytes, but this cell-free source of activity was only apparent if it was tested for colony formation in the presence of added PGE1. The release and action of the PGE1 induced T-lymphocyte erythroid enhancing activity did not require the presence of serum. Erythroid colony formation by NALT- bone marrow cells was not enhanced by PGE1 alone, by medium conditioned by T-lymphocytes in the absence of PGE1, or by PGE1 plus medium conditioned by T-lymphocytes in the absence of PGE1. The results suggest that the PGE1 enhancement of erythroid colony formation occurs by an apparently synergistic action on non-T-lymphocytes by PGE1 itself and by a factor or factors released from T8+ lymphocytes in response to PGE1.