The AML1/ETO Target LAT2 Membrane Adaptor Molecule Is Regulated during Normal Monocytic Differentiation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2401-2401
Author(s):  
Jesus Duque-Afonso ◽  
Leticia Solari ◽  
Michael Luebbert
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Lines ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Gm Csf ◽  
Adaptor Molecule ◽  
Cd34 Cells ◽  
Conditional Expression

Abstract LAT2 (NTAL/LAB/WBSCR5) is a 28 KDa membrane protein which acts as adaptor molecule in the signalling pathways of FcεR I, c-Kit, B cell and T cell receptor. Bone marrow-derived mast cells from knock-out (KO) mice are hyperresponsive to stimulation via FcεR I. Although LAT2 is highly expressed in B cells, no major changes were found in function or development of B cells from LAT2 KO mice. An autoimmunity syndrome in LAT2 KO mice is caused, at least in part, by hyperreactivity and higher proliferation of T cells. Previously, we showed that LAT2 mRNA is repressed in vivo by AML1/ETO which was confirmed by others in several large series of primary AML blasts. We wished to elucidate the possible role of LAT2 during the myelopoiesis. AML1/ETO was induced by Ponasterone A in an ecdysone-inducible system in U937 cells (9/14/18 cell line). AML bone marrow samples from 43 patients (pts) were analyzed for LAT2 expression. Several myeloid cell lines were treated either with ATRA, DMSO or PMA for 3 days. Normal CD34+ cells were differentiated ex vivo by G-CSF towards granulocytes and by GM-CSF plus IL-4 towards monocytes and dendritic cells. LAT2 expression was determined by Northern and Western blot. LAT2 protein was repressed not only in AML1/ETO positive primary AML blasts (6/6), but also in blasts from patients with deletions of chromosome 7 (3/4) and the t(15;17) (4/4); expression was moderate to high in AML blasts with normal karyotype (14/15). LAT2 was expressed in normal monocytes and even higher in alveolar macrophages but not in granulocytes of healthy donors. It was downregulated after ATRA-induced granulocytic differentiation of NB4, HL60 and U937 cells but upregulated after DMSO-induced granulocytic differentiation of HL60 cells and PMA-induced monocytic-macrophage differentiation of HL60, U937 and Kasumi-1 cells. In normal CD34+ cells, LAT2 was strongly induced 7 days after the addition of G-CSF and GM-CSF+IL4 respectively, but after 14 days it was downregulated (0.7 +/− 0.4-fold) by G-CSF-induced granulocytic differentiation and upregulated (5.8 +/− 2.8-fold) by GM-CSF+IL4-induced monocytic-DC differentiation. Conditional expression of AML1/ETO in 9/14/18-U937 cells partially inhibited the PMA- and vitamin D3-induced monocytic differentiation of these cells, as determined by FACS for CD11b and CD11c. In conclusion, LAT2 protein is strongly repressed by AML1/ETO in primary leukemias and is upregulated during the monocytic differentiation in several cell lines and normal CD34+ cells. Further studies in a LAT2 knock-down by shRNAs in U937 cells are warranted to functionally address its possible role in monocytic differentiation.

Inhibition of TET2 Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine Disturbs Myelopoiesis and Granulo-Monocytic Differentiation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1584-1584 ◽  
Author(s):  
Elodie Pronier ◽  
Carole Almire ◽  
Barbara da Costa Reis Monte Mor ◽  
Audrey Simon ◽  
Aline Massé ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cord Blood ◽  
Cell Line ◽  
Cell Lines ◽  
Umbilical Cord Blood ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Monocytic Cells ◽  
Knock Down ◽  
Cd34 Cells

Abstract Abstract 1584 TET2, a gene frequently mutated in myeloid malignancies, encodes an oxygenase that may convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in hematopoietic cells. Using high performance liquid chromatography coupled to tandem mass sprectrometry (HPLC-MS-MS), we found lower levels of 5hmC in erythroblasts from patients with myeloproliferative neoplasms and TET2 mutations than in erythroblasts from non mutated patients. To study the function of TET2 during myelopoiesis, we used lentiviruses expressing short hairpin RNA (shRNA) designed to inhibit TET2 expression in CD34+ cells from normal bone marrow or umbilical cord blood. TET2 mRNA was knocked down by 56 to 74% in several human leukemia cell lines expressing shRNA-TET2, compared to cell lines expressing scramble shRNA. TET2 expression was then assessed in these cell lines by western blot, which showed a 49% reduction of TET2 protein in TF1 cell line, and 80 to 90% reduction in Kasumi, Uke1, and Mo7E cell lines. In addition, knock down of TET2 led to a 73% decrease in 5hmC in Mo7E cell line DNA whereas 5mC and cytosine contents remained unchanged as measured by HPLC-MS-MS. These results indicate that TET2 has a role in the hydroxylation of 5mC in human cells of hematopoietic origin. We then studied the consequences of TET2 knock-down in umbilical cord blood CD34+ cells in vitro. We observed a skewing of CD34+CD38- progenitor differentiation toward the myeloid lineage (52 +/− 4% of the TET2 knock down cells versus 35 +/− 3% of the control cells, n=3, p=0.001) at the expense of lympho-myeloid development and B cell and natural killer (NK) lymphoid lineages in a B/myeloid/NK culture condition. Methylcellulose in the presence of EPO, IL3, SCF, G-CSF showed greater numbers of CFU-G/GM (66 +/− 4 versus 58 +/− 3 colonies per 1000 input CD34+ cells) and lower numbers of BFU-E (60 +/−19 versus 78 +/− 19) in TET2 knock-down samples than in control samples (n= 4, p<0.05) indicating that there was also a skewing toward the granulo-monocytic differentiation at the expense of the erythroid lineage. In addition, in presence of G-CSF, IL3, and FLT3-ligand granulocytic differentiation was delayed in TET2 knock-down cells with a relative excess of monocytic cells at day 10 of culture as assessed by the analysis of cell morphology (47 +/− 4% monocytic cells versus 37 +/− 3%, n=5, p=0.006) and immunophenotype (52 +/− 3% of CD14+ cells versus 39+/−8%, n=5, p=0.002). We then analyzed the expression of a set of transcription factors at the mRNA level that confirmed that some transcription factors specific to granulocytic differentiation were under-expressed in TET2 knocked down cells between day 5 and day 10 of culture. Together, our results show that TET2 participates to the conversion of 5mC to 5hmC in hematopoietic cells, and suggest that TET2 inactivation may have a role in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.

JAK2-Mediated Extrinsic Survival of CML Stem Cells: Exploring the Potential Combination of BCR-ABL and JAK2 Inhibitors In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 199-199
Author(s):  
Elie Traer ◽  
Ryan MacKenzie ◽  
Jennifer Snead ◽  
Brian J Druker ◽  
Michael W Deininger
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Median Survival ◽  
Conditioned Media ◽  
High Dose ◽  
Protective Effects ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Jak2 Inhibitors

Abstract Abstract 199 Background: The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are very effective for the treatment of chronic phase CML. However, the majority of these patients continue to have persistence of CML cells despite continued therapy, suggesting that TKIs fail to target leukemic stem cells (LSCs). There is increasing evidence that the bone marrow microenvironment provides a sanctuary to LSCs, thereby contributing to persistence. Results: We used the human stromal cell lines (HS-5, HS-23, HS-27a) to model the microenvironment. Conditioned media from HS-5, but not HS-23 or HS-27a, reduced apoptosis of CML cell lines treated with TKIs (K562, LAMA-84, KBM-5 and KYO-1), consistent with previous reports. Similarly, CML CD34+ cells were protected from 5 μ M imatinib in a 4 day co-culture with HS-5 cells, as assessed by CFU-GM colony survival (23% vs 9% when compared to untreated controls, N=6, p=0.018). We were also able to demonstrate protection from TKIs with transwells over HS-5 and with HS-5 conditioned media, which suggests that factors secreted by HS-5 cells protect CML cells from TKIs. Cytokine analysis of conditioned media revealed relatively higher concentrations of IL6, IL-8, MCP-1, MCP-3, G-CSF and GM-CSF from HS-5 as compared to HS-23 and HS-27a. Since IL-6, G-CSF and GM-CSF are known to signal via JAK2, we tested combinations of imatinib and JAK2 inhibitors (TG101209 or CYT387) using our in vitro assay. Combination treatment with imatinib and CYT387 or TG101209 abrogated the protective effects of HS-5 conditioned media in CML cell lines. Combination treatment of CML CD34+ in HS-5 co-culture assays also abrogated the protective effects of stroma on colony formation. However, we observed that both normal CD34+ and CML CD34+ colony formation was dramatically reduced by JAK2 inhibitors using our HS-5 co-culture system, particularly at higher doses. Thus, it was unclear if a potential therapeutic window existed in vivo. To test the potential of combination therapy in vivo, we infected marrow from Balb/c mice with a retrovirus that simultaneously expresses BCR-ABL and GFP, followed by transplantation into lethally irradiated syngeneic recipients. The mice were separated into five cohorts: vehicle control, TG101209 (200mg/kg/d), nilotinib (75mg/kg/d), nilotinib + low-dose TG101209 (50mg/kg/d) and nilotinib + high-dose TG101209 (200mg/kg/d). The vehicle-treated control group died rapidly of myeloproliferative disease (MPD) with a median survival of 15.5 days. The median survival of mice treated with TG101209 was slightly prolonged at 20.5 days (p=0.06); however, these mice also died of MPD with enlarged spleens/livers and lung hemorrhage. The survival curves of mice treated with nilotinib monotherapy and nilotinib + low-dose TG101209 were similar (median survival not reached at termination of experiment). Mice treated with nilotinib + high-dose TG101209 initially had minimal mortality, however on day 26 the mice began to die without signs of MPD (no definitive cause of death at autopsy) and thus the remaining cohort was sacrificed on day 27 for analysis. In stark contrast to the other cohorts, the spleens of these mice were very small, leukopenic, and largely devoid of normal follicles, with decreased spleen weight compared to mice treated with nilotinib monotherapy (0.025 vs. 0.072 gm, p<0.01). The bone marrow was also profoundly hypocellular, suggesting that myelosuppresion (anemia and leukopenia in particular) may have been a factor in mortality. However, despite toxicity this combination may retain a degree of selectivity for BCR-ABL cells since disease burden, as measured by the percentage of GFP-positive cells, was reduced compared to nilotinib monotherapy (spleen: 4% vs 11.7%, p=0.047; bone marrow: 8.7% vs 13.8%, p=0.22). Conclusions: (1) Factors secreted by human bone marrow stromal cells attenuate the effects of imatinib in CML cell lines and primary CML CD34+ cells in a JAK2-dependent fashion. (2) Simultaneous in vivo inhibition of BCR-ABL and JAK2 dramatically reduces BCR-ABL expressing cells, but at the cost of marrow toxicity. We speculate that this limitation may be overcome by intermittent rather than continuous JAK2 inhibition, a strategy that might avoid toxicity while reducing persistent BCR-ABL disease burden. Disclosures: Deininger: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Genzyme: Research Funding; Ariad: Consultancy.

scholarly journals Downregulation of c-Jun Expression by Transcription Factor C/EBPα Is Critical for Granulocytic Lineage Commitment

2002 ◽  
Vol 22 (24) ◽  
pp. 8681-8694 ◽  
Author(s):  
Janki Rangatia ◽  
Rajani Kanth Vangala ◽  
Nicolai Treiber ◽  
Pu Zhang ◽  
Hanna Radomska ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Knockout Mouse ◽  
Leucine Zipper ◽  
Fetal Liver ◽  
Mrna Level ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Conditional Expression ◽  
Factor C ◽  
Transcription Factor Complex

ABSTRACT The transcription factor C/EBPα is crucial for the differentiation of granulocytes. Conditional expression of C/EBPα triggers neutrophilic differentiation, and C/EBPα can block 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation of bipotential myeloid cells. In C/EBPα knockout mice, no mature granulocytes are present. A dramatic increase of c-Jun mRNA in C/EBPα knockout mouse fetal liver was observed. c-Jun, a component of the AP-1 transcription factor complex and a coactivator of the transcription factor PU.1, is important for monocytic differentiation. Here we report that C/EBPα downregulates c-Jun expression to drive granulocytic differentiation. An ectopic increase of C/EBPα expression decreases the c-Jun mRNA level, and the human c-Jun promoter activity is downregulated eightfold in the presence of C/EBPα. C/EBPα and c-Jun interact through their leucine zipper domains, and this interaction prevents c-Jun from binding to DNA. This results in downregulation of c-Jun's capacity to autoregulate its own promoter through the proximal AP-1 site. Overexpression of c-Jun prevents C/EBPα-induced granulocytic differentiation. Thus, we propose a model in which C/EBPα needs to downregulate c-Jun expression and transactivation capacity for promoting granulocytic differentiation.

Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages

1990 ◽  
Vol 10 (7) ◽  
pp. 3562-3568
Author(s):  
M Principato ◽  
J L Cleveland ◽  
U R Rapp ◽  
K L Holmes ◽  
J H Pierce ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Hematopoietic Differentiation ◽  
Developmental Relationships ◽  
Murine Bone Marrow ◽  
B Lineage ◽  
Marrow Cells

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.

scholarly journals Hepatocyte Growth Factor Is Constitutively Produced by Human Bone Marrow Stromal Cells and Indirectly Promotes Hematopoiesis

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1560-1565 ◽  
Author(s):  
Kenji Takai ◽  
Junichi Hara ◽  
Kunio Matsumoto ◽  
Gaku Hosoi ◽  
Yuko Osugi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Gm Csf ◽  
Tumor Growth Factor ◽  
Cd34 Cells ◽  
Factor Α ◽  
Hepatocyte Growth

Bone marrow (BM) stromal cells are required for normal hematopoiesis. A number of soluble factors secreted by these cells that mediate hematopoiesis have been characterized. However, the mechanism of hematopoiesis cannot be explained solely by these known factors, and the existence of other, still unknown stromal factors has been postulated. We showed that hepatocyte growth factor (HGF ) is one such cytokine produced by human BM stromal cells. BM stromal cells were shown to constitutively produce HGF and also to express the c-MET/HGF receptor. The production of HGF was enhanced by addition of heparin and phorbol ester. Dexamethasone and tumor growth factor-β (TGF-β) inhibited the production of HGF. Interleukin-1α (IL-1α) tumor necrosis factor-α (TNF-α), and N6,2′-o-dibutyryl-adenosine-3′:5′-cyclic monophosphate (dbc-AMP) showed no obvious influence on HGF production. Western blot analysis of HGF derived from BM stromal cells showed two bands at 85 and 28 kD corresponding to native and variant HGF, respectively. Addition of recombinant HGF significantly promoted the formation of burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte erythroid macrophage (CFU-GEM) by BM mononuclear cells in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF ), but the formation of CFU-GM was not modified. However, HGF had no effects on colony formation by purified CD34+ cells. Within BM mononuclear cells, c-MET was expressed on a proportion of cells (CD34−, CD33+, CD13+, CD14+, and CD15+), but was not found on CD34+ cells. We conclude that HGF is constitutively produced by BM stromal cells and that it enhances hematopoiesis. In addition, expression of c-MET on the stromal cells suggests the presence of an autocrine mechanism, operating through HGF, among stromal cells.

scholarly journals Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3375-3383 ◽  
Author(s):  
T Tsujimoto ◽  
IA Lisukov ◽  
N Huang ◽  
MS Mahmoud ◽  
MM Kawano
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
B Cell Survival

By using two-color phenotypic analysis with fluorescein isothiocyanate- anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM- 102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein- 1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre- B cells in the presence of IL-7, but coculture of plasma cells with KM- 102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF- beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL- 7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.

scholarly journals Platelet Factor 4 and Other CXC Chemokines Support the Survival of Normal Hematopoietic Cells and Reduce the Chemosensitivity of Cells to Cytotoxic Agents

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2328-2335 ◽  
Author(s):  
Zhong Chao Han ◽  
Min Lu ◽  
Junmin Li ◽  
Mai Defard ◽  
Bernadette Boval ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Cell Lines ◽  
Cancer Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Platelet Factor 4 ◽  
Cytotoxic Agents ◽  
Platelet Factor ◽  
Cd34 Cells

Abstract The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 μg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 μg/mL. PF4 also supported the viability of CD34+ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor β1 (TGFβ1) and cytotoxic drugs. Pretreatment of CD34+ cells by PF4, but not by TGFβ1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34+ cells were preincubated with PF4 or TGFβ1 for 12 days in hematopoietic growth factor–rich medium, an increased number of remaining CD34+ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC50) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 μg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU–granulocyte/macrophage (CFU-GM), and burst-forming units–erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 μg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.

scholarly journals Regulation of the human cellular glutathione peroxidase gene during in vitro myeloid and monocytic differentiation

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3902-3908 ◽  
Author(s):  
Q Shen ◽  
S Chada ◽  
C Whitney ◽  
PE Newburger
Keyword(s):  
Steady State ◽  
Glutathione Peroxidase ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Dimethyl Formamide ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Regulation Of Expression

We have used the HL-60 and PLB-985 myeloid leukemia cell lines to examine the regulation of expression of the important intracellular antioxidant enzyme, glutathione peroxidase (GSH-Px), during phagocytic cell differentiation in vitro. Induction of differentiation along the monocytic pathway by phorbol ester results in an approximately twofold rise in enzyme activity and a parallel increase in the rate of 75Se incorporation into immunoprecipitable GSH-Px protein. Induction along the granulocytic pathway by dimethyl formamide (DMF) results in similar changes in steady-state enzyme levels and rates of GSH-Px protein synthesis. Steady-state levels of GSH-Px gene transcripts also increase more than twofold, approximately in parallel with the enzyme levels. Nuclear run-on transcription assays of GSH-Px mRNA synthesis show ratios of induced to uninduced transcript levels of 2.24 and 1.59 with phorbol myristate acetate (PMA) induction and DMF, respectively, in HL- 60 cells, and ratios of 1.34 and 3.46 with PMA and DMF, respectively, in PLB-985 cells. Half lives of GSH-Px mRNA are unchanged or slightly shorter after differentiation of HL-60 cells, and slightly longer after induction of PLB-985. Overall, the present studies show that GSH-Px activity rises during in vitro-induced monocytic or granulocytic differentiation of myeloid cell lines and that the increased expression of the cellular GSH-Px gene occurs through complex mechanisms that include transcriptional up-regulation. This pattern contrasts with the nearly complete cotranslational regulation of GSH-Px expression by exogenous selenium.

Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 110-114 ◽  
Author(s):  
I McNiece ◽  
R Andrews ◽  
M Stewart ◽  
S Clark ◽  
T Boone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Synergistic Interaction ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.

scholarly journals Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.

1995 ◽  
Vol 181 (5) ◽  
pp. 1805-1815 ◽  
Author(s):  
J P Lévesque ◽  
D I Leavesley ◽  
S Niutta ◽  
M Vadas ◽  
P J Simmons
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Normal Bone Marrow ◽  
Gm Csf ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Late Antigen ◽  
Beta 1 Integrin ◽  
Functional States ◽  
Adhesive Interactions

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine-dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.

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