Biomedical subjects

R Delwel

Publications and source records attributed to R Delwel.

At least 19 recordsLinked to original sources

PubMed2005

BCR/ABL-mediated downregulation of genes implicated in cell adhesion and motility leads to impaired migration toward CCR7 ligands CCL19 and CCL21 in primary BCR/ABL-positive cells.

The mechanism underlying p210(BCR/ABL) oncoprotein-mediated transformation in chronic myelogenous leukemia (CML) is not fully understood. We hypothesized that p210(BCR/ABL) suppresses expression of genes which may explain at least some of the pathogenetic features of CML. A subtractive cDNA library was created between BCR/ABL-enhanced-green-fluorescent-protein (GFP)-transduced umbilical cord blood (UCB) CD34+ cells and GFP-transduced UCB CD34+ cells to identify genes whose expression is downregulated by p210(BCR/ABL). At least 100 genes were identified. We have confirmed for eight of these genes that expression was suppressed by quantitative real-time-RT-PCR (Q-RT-PCR) of additional p210(BCR/ABL)-transduced CD34+ UCB cells as well as primary early chronic phase (CP) bone marrow (BM) CML CD34+ cells. Imatinib mesylate reversed downregulation of some genes, to approximately normal levels. Several of the genes are implicated in cell adhesion and motility, including L-selectin, intercellular adhesion molecule-1 (ICAM-1), and the chemokine receptor, CCR7, consistent with the known defect in adhesion and migration of CML cells. Compared with GFP UCB or normal (NL) BM CD34+ cells, p210 UCB and CML CD34+ cells migrated poorly towards the CCR7 ligands, CCL19 and CCL21, suggesting a possible role for CCR7 in the abnormal migratory behavior of CML CD34+ cells.

Cell Adhesion↗

PubMed2000

Phenotyping of Evi1, Evi11/Cb2, and Evi12 transformed leukemias isolated from a novel panel of cas-Br-M murine leukemia virus-infected mice.

Cas-Br-M murine leukemia virus (MuLV) is a slow-transforming retrovirus that potently induces leukemias in mice and therefore is well suited for retroviral insertional mutagenesis. We used Cas-Br-M MuLV in NIH/Swiss mice to establish a new panel of mainly myeloid leukemias. All tumors found in leukemic animals were classified by gross pathology, morphology, and immunophenotype, as well as the incidence of known common virus integration sites (VISs) in MuLV-induced myeloid malignancies (i.e., Evi1, Evi11/Cb2, Evi12, Fli1, and c-Myb). Interestingly, male mice were more susceptible than females to the induction of leukemia by Cas-Br-M MuLV. Seventy-four of the Cas-Br-M MuLV-inoculated mice developed a severe splenomegaly, sometimes in association with a thymoma. Although most of the immunophenotyped Cas-Br-M MuLV tumors were of myeloid origin (58%), numerous T-cell leukemias (21%) and mixed myeloid/T-cell leukemias (21%) were found. The myeloid leukemias and myeloid compartment of the mixed leukemias were further characterized by immunophenotyping with stem cell-, myeloid-, and erythroid-specific antibodies. The known Cas-Br-M MuLV common VISs (Evi1, Evi11/Cb2, and Evi12) were demonstrated in 19%, 12%, and 20% of the cases, respectively, whereas no Fli1 and c-Myb rearrangements were found. Integrations into Evi1 were restricted to myeloid leukemias, whereas those in Evi11/Cb2 and Evi12 were identified in myeloid as well as T-lymphoid leukemias. This panel of well characterized Cas-Br-M MuLV-induced hematopoietic tumors may be useful for the isolation and characterization of new proto-oncogenes involved in myeloid or T-cell leukemias.

3T3 Cells↗

PubMed2000

Erythroid defects and increased retrovirally-induced tumor formation in Evi1 transgenic mice.

Aberrant expression of the Evi1 (ecotropic virus integration site 1) proto-oncogene has been associated with hematopoietic malignancies in both mice and man. To determine the effect of enforced expression of Evi1 in vivo, we developed a transgenic mouse model utilizing the murine Sca-1 (Ly-6E.1) promoter. Here, we describe the generation and analysis of three independent lines of Evi1 transgenic mice. Transgenic animals of two founder lines developed normally. These mice did not show any obvious hematological abnormalities but showed a significant reduction in the number of bone marrow colony-forming unit erythroid (CFU-E)-derived colonies. This implies a defect of normal erythroid hematopoiesis affecting relatively late erythroid progenitor cells. We also show that when newborn Evi1 transgenic mice of these two lines were infected with Cas-Br-M MuLV, tumor incidence was greatly enhanced in comparison with nontransgenic littermates, indicating an increased susceptibility for leukemia development. Interestingly, analysis of a third founder line revealed that all male progeny consistently displayed severely impaired erythropoiesis with major defects in the bone marrow, spleen and peripheral blood. Taken together, our results present the first evidence of Evi1 disturbing normal erythropoiesis in vivo and provides evidence for cooperative potential of Evi1 in tumor progression.

Animals↗

PubMed2000

Enhancement of erythropoietin-stimulated cell proliferation by Anandamide correlates with increased activation of the mitogen-activated protein kinases ERK1 and ERK2.

INTRODUCTION: Anandamide (ANA) is an endogenous ligand for the cannabinoid receptors Cb1 and Cb2 that is able to synergistically stimulate the proliferation of hematopoietic growth factor-dependent blood cells in serum-free culture. To elucidate the mechanisms by which ANA enhances the proliferative responses of hematopoietic cells, we investigated the ANA-mediated effects on proliferation, cell cycling, apoptosis and intracellular signaling of erythropoietin-stimulated 32D/EPO cells. MATERIALS AND METHODS: 32D/EPO cells were cultured serum free to determine the effects of EPO and anandamide on these cells. Proliferation was analyzed by tritiated thymidine incorporation. Apoptosis as well as cell cycle analysis was carried out by flow cytometry. MAPKinase activation was determined by Western blotting, using phospho-specific MAPK antibodies. RESULTS: Simultaneous addition of erythropoietin (EPO) and ANA enhanced DNA synthesis and increased 32D/EPO cell numbers in serum-free culture. Interestingly, ANA did not alter the G1/S transition but it accelerated each of the successive cell cycle phases of EPO-stimulated 32D/EPO cells. Percentages of apoptotic 32D/EPO cells were equally low in cultures supplemented with EPO alone or a combination of EPO and ANA. Both cultures showed enhanced activation of two mitogen-activated protein kinases, namely, extracellular factor responsive kinases 1 and 2 (ERK1/2), as well as the MAPK-target gene protein c-Fos. This fully correlated with the synergistic stimulation of proliferation of 32D/EPO cells by EPO and ANA. ANA had no effect on EPO-induced STAT-5 activation of 32D/EPO cells. Experiments with the Cb2 receptor-specific antagonist SR144528 demonstrated that the synergistic stimulation of proliferation by ANA was partially Cb2 receptor-mediated. CONCLUSION: These data suggest that the positive effects of ANA on the erythropoietin-induced proliferation of 32D/EPO cells are mediated by receptor-dependent as well as receptor-independent mechanisms, both of which involve activation of the mitogen-activated protein kinases, ERK1/2.

Animals↗

PubMed1999

Retroviral insertions in Evi12, a novel common virus integration site upstream of Tra1/Grp94, frequently coincide with insertions in the gene encoding the peripheral cannabinoid receptor Cnr2.

The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also referred to as Cb2). Here we show that Cnr2 is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions in Evi11 were cloned and shown to be located within the 3' untranslated region of the candidate proto-oncogene Cnr2. These results suggest that proviral insertion in the Cnr2 gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolate Evi11/Cnr2 collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS, Evi12 (14%). Interestingly, 54% of the Evi11/Cnr2-rearranged primary tumors contained insertions in Evi12 as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown that Evi12 resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated that Evi12 is located upstream of the gene encoding the molecular chaperone Tra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22-24. Thus, Tra1/Grp94 is a candidate target gene for retroviral activation or inactivation in Evi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression of Tra1/Grp94. Additional studies are required to determine whether Tra1/Grp94 or another candidate proto-oncogene in Evi12 is involved in leukemogenesis.

3T3 Cells↗

PubMed1998

The peripheral cannabinoid receptor, Cb2, in retrovirally-induced leukemic transformation and normal hematopoiesis.

Following retroviral insertional mutagenesis we recently identified the gene encoding the peripheral cannabinoid receptor (Cb2) near a common virus integration site (VIS), Evi11. In 13 out of 105 Cas-Br-M murine leukemia virus (MuLV) induced leukemias retroviral integrations occured either in the 5' or 3' part of the Cb2 gene. The Cb2 receptor protein is 44% homologous to the central cannabinoid receptor Cb1, which belongs to the superfamily of seven transmembrane (7TM) receptors. Cb1 is mainly expressed in brain, whereas Cb2 encodes the hematopoietic form. Besides the natural cannabinoids, delta9-tetrahydrocannabinol (delta9-THC) and cannabinol, and the many synthetic agonists that have been generated, e.g CP55,940 or WIN55,212-2, several endogenous ligands have recently been identified. These include the arachidonic acid derivatives anandamide and 2-arachidonylglycerol as well as the fatty acid palmitoylethanolamide. Although in the past many studies described growth inhibitory effects of cannabinoid agonists on the in vitro proliferation of hematopoietic cells, recent studies demonstrated that activation of Cb2 may have growth stimulatory effects on blood precursor cells. We demonstrated that many murine hematopoietic growth factor (HGF) dependent cell lines also require the presence of anandamide for optimal growth in serum free culture. Thus, the Cb2 receptor may be an important regulator of normal hematopoietic growth and development. These results strengthen our finding that Cb2 is a proto-oncogene and may implicate a growth advantage for leukemia cells that aberrantly express Cb2. Here we briefly review the mechanisms and application of retroviral insertional mutagenesis in leukemic transformation in mice and discuss the role of the peripheral cannabinoid receptor in leukemia development and normal hematopoiesis.

Animals↗

PubMed1997

A rapid RT-PCR based method to isolate complementary DNA fragments flanking retrovirus integration sites.

Proto-oncogenes in retrovirally induced myeloid mouse leukemias are frequently activated following retroviral insertion. The identification of common virus integration sites (VISs) and isolation of the transforming oncogene is laborious and time consuming. We established a rapid and simple PCR based procedure which facilitates the identification of VISs and novel proto-oncogenes. Complementary DNA fragments adjacent to retrovirus integration sites were selectively isolated by applying a reverse transcriptase (RT) reaction using an oligo(dT)-adaptor primer, followed by PCR using the adaptor sequence and a retrovirus long terminal repeat (LTR) specific primer. Multiple chimeric cDNA fragments suitable for Southern and northern blot analysis were isolated.

Animals↗

PubMed1997

Anandamide, a natural ligand for the peripheral cannabinoid receptor is a novel synergistic growth factor for hematopoietic cells.

We recently demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) may be a proto-oncogene involved in murine myeloid leukemias. We show here that Cb2 may have a role in hematopoietic development. RNAse protection analysis showed that Cb2 is normally expressed in spleen and thymus. Cb2 mRNA is also expressed in 45 of 51 cell lines of distinct hematopoietic lineages, ie, myeloid, macrophage, mast, B-lymphoid, T-lymphoid, and erythroid cells. The effect of the fatty acid anandamide, an endogenous ligand for cannabinoid receptors, on primary murine marrow cells and hematopoietic growth factor (HGF)-dependent cell lines was then investigated. In vitro colony cultures of normal mouse bone marrow cells showed anandamide to potentiate interleukin-3 (IL-3)-induced colony growth markedly. Whereas HGFs alone stimulate proliferation of the various cell lines in serum-free culture only weakly, anandamide enhances the proliferative response of the cell lines to HGFs profoundly. This was apparent for responses induced by IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. Anandamide was already effective at concentrations as low as 0.1 to 0.3 micromol/L and plateau effects were reached at 0.3 to 3 micromol/L. The addition of anandamide as single growth factor had no effect. The costimulatory effect of anandamide was not evident when cells were cultured with fetal calf serum (FCS), suggesting that FCS contains anandamide or another ligand capable of activating the peripheral cannabinoid receptor. Other cannabinoid ligands did not enhance the proliferative responsiveness of hematopoietic cells to HGFs. Transfection experiments of Cb2 in myeloid 32D cells showed that anandamide specifically activates proliferation through activation of the peripheral cannabinoid receptor. Anandamide appears to be a novel and synergistic growth stimulator for hematopoietic cells.

Animals↗

PubMed1997

Characterization of the C3 YAC contig from proximal mouse chromosome 17 and analysis of allelic expression of genes flanking the imprinted Igf2r gene.

The imprinted mouse insulin-like growth factor type 2 receptor (Igf2r) maps to the middle of a gene-rich region in band A2 of mouse chromosome 17. The t(Lub2) chromosome 17 variant contains a small deletion that removes at least seven genes including Igf2r. We have constructed a YAC contig spanning the entire t(Lub2) deletion and created a restriction map that covers 700 kb. The position, transcription orientation, and imprinted status of the genes immediately flanking Igf2r have been assessed. We show here that the Mas gene, which lies 65 kb upstream to Igf2r, contains a novel 5' exon and is not imprinted in adult tissues. We further show that the recently identified Lx1 gene lies immediately downstream and is also expressed from both parental alleles in adult tissues. The remaining genes in this region have previously been shown to be biallelically expressed.

Alleles↗

PubMed1997

The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11.

A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.

3T3 Cells↗

PubMed1995

The Evi-1 zinc finger myeloid transforming protein binds to genomic fragments containing (GATA)n sequences.

The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.

Animals↗

PubMed1993

Granulocyte-macrophage colony-stimulating factor receptors alter their binding characteristics during myeloid maturation through up-regulation of the affinity converting beta subunit (KH97).

Acute myeloid leukemia blasts express dual affinity (high and low) granulocyte-macrophage colony-stimulating factor (GM-CSF) binding, and the high affinity GM-CSF binding is counteracted by excess interleukin-3 (IL-3). Neutrophils express a single class of GM-CSF-R with intermediate affinity that lack IL-3 cross-reactivity. Here we demonstrate the differentiation associated changes of GM-CSF binding characteristics in three models representative of different stages of myeloid maturation. We find that high affinity GM-CSF binding is converted into intermediate affinity binding, which still cross-reacts with IL-3, beyond the stage of promyelocytes. During terminal maturation towards neutrophils, IL-3 cross-reactivity is gradually lost. We sought to determine the mechanism underlying the affinity conversion of the GM-CSF-R. Northern and reverse transcriptase-polymerase chain reaction analysis of GM-CSF-R alpha and -beta c (KH97) transcripts did not provide indications for the involvement of GM-CSF-R splice variants in the formation of the intermediate affinity GM-CSFR complex. In COS-cell transfectants with increasing amounts of beta c in the presence of a fixed number of GM-CSF-R alpha chains, the high affinity GM-CSF binding converted into intermediate affinity GM-CSF binding. These results are discussed in view of the concept that increasing expression of beta c subunits may cause alternative oligomerization of the GM-CSF-R alpha and -beta c subunits resulting in the formation of intermediate rather than high affinity GM-CSFR alpha.beta c complexes.

Acute Disease↗

PubMed1993

Autonomous proliferation of leukemic cells in vitro as a determinant of prognosis in adult acute myeloid leukemia.

BACKGROUND AND METHODS: A characteristic of acute myeloid leukemia is the frequent ability of the leukemic cells to sustain their own proliferation in vitro. To determine the clinical importance of this property, we measured the uptake of tritiated thymidine by leukemic cells in serum-free and cytokine-free cultures as a means of determining the rate of spontaneous proliferation in 114 patients with newly diagnosed acute myeloid leukemia. Proliferation was then classified according to three quantitative levels of activity and related to overall survival and to treatment outcome (the response to treatment, the actuarial probability of relapse, and disease-free survival) in 91 patients who were treated with chemotherapy to induce remission. RESULTS: Of the 114 patients, 37 had low, 39 had intermediate, and 38 had high levels of proliferation. The probability of survival at three years was 36 percent among patients with low levels of proliferative activity and 3 percent among those with high levels (P < 0.001). Among the patients treated with chemotherapy, those with low rates of proliferative activity had a 68 percent rate of complete remission and a 49 percent probability of remaining free of relapse, whereas those with high rates of proliferative activity had only a 39 percent rate of complete remission (P = 0.04) and an 11 percent probability of remaining in complete remission (P = 0.009). The probability of disease-free survival at three years among the patients in complete remission after chemotherapy was 49 percent among those with low rates of proliferative activity and 9 percent among those with high rates (P = 0.004). Accordingly, patients with low rates of proliferative activity also had a significantly higher rate of overall survival (44 percent vs. 4 percent; P = 0.002). Patients whose cells had intermediate levels of proliferation in vitro had intermediate rates of survival, relapse, and disease-free survival. CONCLUSIONS: The capacity of leukemic blasts for autonomous proliferation is associated with highly aggressive acute myeloid leukemia.

Acute Disease↗

PubMed1993

Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA.

Expression of the Evi-1 gene is activated in murine myeloid leukemias by retroviral insertions and in human acute myelogenous leukemia by translocations and inversions involving chromosome band 3q26 where the gene resides. Aberrant expression of the Evi-1 gene has been shown to interfere with myeloid differentiation, which is proposed to be the basis for its role in leukemias. The Evi-1 gene encodes a 145-kDa DNA-binding protein containing two domains of seven and three Cys2-His2 zinc fingers. Previous studies identified a portion of the consensus DNA-binding sequence for the first domain of zinc fingers. The experiments presented here extend these studies and demonstrate that the first domain recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA. The first three fingers of the first domain do not detectably bind DNA but contribute to the binding by conferring a relative specificity for GACAA verses GATAA in the first position. The first three fingers also contribute to optimal binding of the 15-nucleotide consensus sequence.

Amino Acid Sequence↗

PubMed1993

Effects of mast cell growth factor on acute myeloid leukemia cells in vitro: effects of combinations with other cytokines.

We have investigated the stimulative effects of mast cell growth factor (MGF) in primary acute myeloid leukemia (AML) in vitro. MGF stimulated DNA synthesis of purified leukemic blasts in eight out of 10 cases and colony formation in four cases in serum-free (SF) culture. MGF synergized with interleukin-3 (IL-3; four out of 10 cases), granulocyte-macrophage colony-stimulating factor (GM-CSF; three out of 10 cases), granulocyte colony-stimulating factor (G-CSF; six out of 10 cases), macrophage colony-stimulating factor (M-CSF; one out of 10 cases) and erythropoietin (EPO; one out of 10 cases) when added to culture in combination. Synergistic effects of MGF in combination with other CSFs were also seen in the colony assay. Antibodies against GM-CSF, M-CSF, G-CSF, and IL-6 did not inhibit the MGF response, suggesting that the stimulative effect of MGF was not mediated through autocrine release of those cytokines. Cell recovery data in liquid cultures that contained MGF, IL-3, or MGF + IL-3, indicated that both MGF and IL-3 augmented the maintenance of clonogenic cells as compared to nonsupplemented cultures, but the effect of the combination of IL-3 + MGF did not show synergy. In contrast, activation of DNA synthesis by MGF was abrogated in the presence of tumor necrosis factor (TNF; four out of 10 cases) and interleukin-4 (IL-4; two out of 10 cases). Fluorescence-activated cell sorting (FACS) analysis with anti c-kit antibodies revealed MGF receptor expression in eight out of nine cases, often in a subpopulation of the cells. Scatchard analysis of MGF receptors in two cases indicated the presence of 1460 and 41,500 (mean) binding sites, respectively, of high affinity (Kd 40-160 pmol/l). The MGF dose-response curve in the presence of IL-3 or GM-CSF resulted in a higher plateau of DNA synthesis, however no shift in the dose response was apparent. The respective reciprocal dose response relations to GM-CSF, IL-3, or G-CSF were similarly elevated when MGF was added. MGF did not alter IL-3 and GM-CSF receptor expression, nor did IL-3, GM-CSF, G-CSF, TNF, or IL-4 influence MGF binding to AML cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute Disease↗

PubMed1992

Involvement of tumor necrosis factor (TNF) receptors p55 and p75 in TNF responses of acute myeloid leukemia blasts in vitro.

Tumor necrosis factor (TNF)-alpha and TNF-beta have multiple effects on human acute myeloid leukemia (AML) cells in vitro, including (1) synergistic stimulation of proliferation with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and upregulation of interleukin-3 (IL-3) and GM-CSF receptors; (2) inhibition of granulocyte-CSF (G-CSF)-induced growth and rapid downmodulation of G-CSF receptors; and (3) induction of autocrine growth. Recently, two distinct TNF receptors (TNF-Rs), TNF-R(p55) and TNF-R(p75), have been identified. In this study, we show that both receptor types may be expressed by AML blasts. It has been investigated whether the different effects of TNF on AML blasts can be explained by differential activation of the distinct TNF-R structures. For this purpose, we used the monoclonal antibodies HTR-1 and HTR-9, specifically recognizing TNF-R(p55), and UTR-1, specific for TNF-R(p75). TNF-(alpha and -beta) mediated synergistic activation with IL-3/GM-CSF, upregulation of IL-3/GM-CSF receptors, inhibition of G-CSF-induced growth, and rapid downmodulation of G-CSF receptors exclusively result from activation of TNF-R(p55). In certain cases in which TNF-alpha, rather than TNF-beta, induces AML growth through an autocrine mechanism, both TNF-R(p55) and (p75) are involved. These data indicate that the variety of TNF responses observed in AML can only be partially explained by differential activation of the TNF-R(p55) and (p75) structures, and that TNF-R(p55) on AML blasts can transduce both positive (synergism with IL-3/GM-CSF) and negative regulatory signals (inhibition of G-CSF-induced proliferation) following TNF activation.

Acute Disease↗

PubMed1991

Tumor necrosis factor regulates the expression of granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors on human acute myeloid leukemia cells.

Tumor necrosis factor (TNF) acts as a potent enhancer of granulocyte-macrophage colony-stimulating factor (GM-CSF)- and interleukin-3 (IL-3)-induced human acute myeloid leukemia (AML) growth in vitro. We have analyzed the effects of TNF alpha on the expression of GM-CSF and IL-3 receptors on AML cells. Incubation of blasts from seven patients with AML in serum-free medium with TNF (10(3) U/mL) and subsequent binding studies using 125I-GM-CSF and 125I-IL-3 show that TNF increases the specific binding of GM-CSF (30% to 280%) and IL-3 (40% to 600%) in all cases. From Scatchard plot analysis it appears that TNF upregulates (1) low-affinity GM-CSF binding sites, (2) common high-affinity IL-3/GM-CSF binding sites, and (3) unique (non-GM-CSF binding) IL-3 binding sites. The effect of TNF is dose dependent and is half maximal at a concentration of 100 U/mL, and becomes evident at 18 hours of incubation with TNF at 37 degrees C, but not at 0 degree C. The GM-CSF dose-response curve of AML-colony-forming units plateaus at a higher level in the presence of TNF, which indicates that additional numbers of cells become responsive to GM-CSF. Incubation of AML blasts with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate or formyl-Met-Leu-Phe (protein kinase C activators) does not influence GM-CSF receptor expression, suggesting that receptor upregulation by TNF is not mediated through activation of protein kinase C. On the other hand, the protein synthesis inhibitor cycloheximide abrogates receptor upregulation induced by TNF. In contrast to these findings in AML, TNF does not upregulate GM-CSF receptor numbers on blood granulocytes or monocytes. We conclude that TNF exerts positive effects on growth factor receptor expression of hematopoietic cells.

Cycloheximide↗

PubMed1991

Induction of granulocytic maturation in acute myeloid leukemia by G-CSF and retinoic acid.

AML cells were cultured free of serum with G-CSF in combination with all-trans-retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyelocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granulocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only.

8-Bromo Cyclic Adenosine Monophosphate↗