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-a has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-a and SCF. Here, we demonstrate that TNF-a potently and directly inhibits SCF-stimulated proliferation of CD34 + hematopoietic progenitor cells. Furthermore, TNF-a 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-a, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNFa were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-a downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well. (J.
Acute promyelocytic leukemia (APL) is a homogeneous subgroup of acute myeloid leukemias (AMLs) characterized by the presence of the t(15,17) translocation and the resulting promyelocytic myeloid leukemia/retinoic acid receptor alpha (PML/RAR alpha) fusion proteins. To date APL is the only AML that is sufficiently sensitive to all-trans retinoic acid's (ATRA) differentiating effect. In vivo ATRA alone achieves complete remission in most APL patients. However, failure or partial responses are observed and the molecular basis of the absence of ATRA response in these patients has not been determined. To gain insights in the cell growth and differentiation of APL cells, expression of hematopoietic growth factors (HGF) shown to be produced by leukemic cells (interleukin-1 beta [IL-1 beta], IL-6, tumor necrosis factor alpha (TNF alpha), granulocyte colony-stimulating factor [G-CSF], granulocyte- macrophage colony-stimulating factor [GM-CSF], and IL-3) was studied in 16 APL samples. Twelve APL cases expressed IL-1 beta, IL-6, and TNF alpha, but not G-CSF, GM-CSF, and IL-3. These cases achieved complete remission with ATRA therapy. The four remaining patients (either TNF alpha negative or G-CSF, GM-CSF or IL-3 positive) did not achieve complete remission with ATRA. In all cases, in vivo response to ATRA therapy was correlated to the in vitro differentiation effect of all- trans retinoic acid 10(-6) mol/L. Thus, ATRA differentiation induction was strongly correlated to the HGF expression (P < .0001). These results suggest that the presence or absence of HGF's expression by APL cells may contribute to the therapeutic effect of ATRA in this disease.
The complex process of neutrophil activation and accumulation is orchestrated by a cascade of cytokines and bioactive lipids produced at the site of inflammation. Neutrophils are a rich source of the potent inflammatory lipid leukotriene B4(LTB4). Granulocyte-macrophage colony-stimulating factor (GM-CSF) can activate neutrophils for an exponential increase in LTB4 production in response to a number of subsequent stimuli. In this report, we examined the temporal regulation, by GM-CSF, of the gene and protein expression of 5-lipoxygenase (5-LOX), a key enzyme of the LTB4 production pathway. Human neutrophils were exposed to 10 ng/mL of GM-CSF for various periods of time and 5-LOX mRNA was measured by Northern blot analysis. We observed no change in 5-LOX mRNA at early time points (0.25 to 3 hours); however, by 18 hours we observed a significant augmentation of 5-LOX-specific message (4.3 +/- 1.7-fold increase; n = 6). Nuclear transcription assays indicated that the rate of 5-LOX gene transcription was augmented threefold in neutrophils incubated with GM-CSF, whereas the half-life of the message was not markedly changed. Parallel experiments indicated that the levels of 5-LOX protein were also increased by GM-CSF. The augmentation was observed within 30 minutes after stimulation and was maximal (5.23 +/- 2.6; n = 4) at 18 hours. Incubation of GM-CSF-stimulated neutrophils with protein synthesis inhibitors resulted in a time-dependent impairment of their ability to produce LTB4, with no inhibition seen during the first hours, a 75% decrease seen by 12 hours, and greater than 95% inhibition seen at 18 hours. Collectively, our data imply that GM-CSF can regulate LTB4 production by two distinct mechanisms: a short-term increase that is not related to increased 5-LOX mRNA expression and is independent of protein synthesis, and a sustained increase in LTB4 production that is associated with the transcriptional activation of the 5-LOX gene, increase in 5-LOX mRNA levels, and dependence on protein synthesis. Such transcriptional modulation of 5-LOX enzyme expression may provide new approaches for therapeutic intervention in protracted inflammatory conditions.
The cell-surface receptor c-kit and its cognate ligand stem-cell factor (SCF) or steel factor (SLF) are important for the maintenance of hematopoiesis both in vitro and in vivo. Transforming growth factor- beta (TGF-beta) has been shown to be a potent inhibitor of SLF-mediated synergistic growth of murine Lin-Sca-1+ progenitor cells, as well as more committed progenitors. In the present study, we examined the regulation of c-kit mRNA and cell-surface expression by TGF-beta. Among the murine hematopoietic progenitor cells tested, the myeloid cell line FDC-P1 and the mast-cell line MC-6, as well as progenitor-enriched bone marrow cells, constitutively expressed functional cell-surface c-kit. Treatment of these progenitor cell lines and primary progenitor cells with TGF-beta resulted in downregulation of cell-surface c-kit expression. This effect was not a secondary event of cell-cycle status. TGF-beta inhibition was dose- and time-dependent, with 50% inhibition seen between 0.3 to 3 ng/mL TGF-beta and maximal inhibition at 30 ng/mL. Using the FDC-P1 cell line, we observed that the inhibition of cell-surface c-kit expression by TGF-beta is preceded by a marked reduction in c-kit mRNA levels starting 2 hours after TGF-beta treatment, and reaches a maximum by 6 hours. The inhibition in steady- state c-kit mRNA levels is explained, in part, by a decrease in the half-life of c-kit transcripts (2 to 4 hours for control cells v 0.5 to 1.5 hours for TGF-beta-treated cells). These findings suggest that TGF- beta regulates the responsiveness of murine hematopoietic progenitors to SLF through a decrease in c-kit message stability leading to decreased cell-surface expression.
Acute promyelocytic leukemia (APL) is a homogeneous subgroup of acute myeloid leukemias (AMLs) characterized by the presence of the t(15,17) translocation and the resulting promyelocytic myeloid leukemia/retinoic acid receptor alpha (PML/RAR alpha) fusion proteins. To date APL is the only AML that is sufficiently sensitive to all-trans retinoic acid's (ATRA) differentiating effect. In vivo ATRA alone achieves complete remission in most APL patients. However, failure or partial responses are observed and the molecular basis of the absence of ATRA response in these patients has not been determined. To gain insights in the cell growth and differentiation of APL cells, expression of hematopoietic growth factors (HGF) shown to be produced by leukemic cells (interleukin-1 beta [IL-1 beta], IL-6, tumor necrosis factor alpha (TNF alpha), granulocyte colony-stimulating factor [G-CSF], granulocyte- macrophage colony-stimulating factor [GM-CSF], and IL-3) was studied in 16 APL samples. Twelve APL cases expressed IL-1 beta, IL-6, and TNF alpha, but not G-CSF, GM-CSF, and IL-3. These cases achieved complete remission with ATRA therapy. The four remaining patients (either TNF alpha negative or G-CSF, GM-CSF or IL-3 positive) did not achieve complete remission with ATRA. In all cases, in vivo response to ATRA therapy was correlated to the in vitro differentiation effect of all- trans retinoic acid 10(-6) mol/L. Thus, ATRA differentiation induction was strongly correlated to the HGF expression (P < .0001). These results suggest that the presence or absence of HGF's expression by APL cells may contribute to the therapeutic effect of ATRA in this disease.
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