Absence Of Granulocyte Colony-stimulating Factor Research Articles

Effect of all-trans retinoid acid and G-CSF on growth, differentiation and RARα2 expression of myeloma cells

To investigate the effect of all-trans retinoid acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) on the growth, apoptosis, differentiation and expression of RARα2 of myeloma cells. Myeloma cell lines OPM2 (RARα2 positive) and U266 (RARα2 negative) were treated with ATRA in the presence or absence of G-CSF. The cells were divided into 6 groups: control groups, G-CSF groups (treated with 1000 U/ml and 2000 U/ml), ATRA groups (treated with 1.0 μmol/L ATRA) and combined groups (treated with 1000 U/mL or 2000 U/mL G-CSF plus 1.0 μmol/L ATRA). The cell viability, growth and apoptosis were examined by MTT method, inverted microscopy and Annexin-V/PI staining, respectively; RARα2 expression was detected by reverse transcription PCR; morphology change was evaluated by Wright-Giemsa staining; CD49e expression were analyzed by flow cytometry. The proliferation of OPM2 cells was inhibited by ATRA treatment (P<0.05) . The growth inhibition rates in combined groups were higher than corresponding single ATRA groups (P<0.05). However, the above effects in U266 cells were not significant (P >0.05). The OPM2 cell stained by Wright-Giemsa in ATRA groups showed that the cell nucleus became smaller, chromatin condensed, number of nucleolus reduced, the volume of cytoplasm increased and the cytoplasm became dark blue. Expression rates of CD49e were low in both U266 and OPM2 cells. Expression of RARα2 in OPM2 cells of combination groups were higher than those of control group and corresponding single groups (P<0.05); and there was no significant difference between control group and G-CSF groups (P>0.05). Expression of RARα2 in U266 cells of control group and G-CSF groups was not detected; and ATRA groups and combination groups had weak expression. ATRA can induce proliferation inhibition in RARα2-expressing myeloma cells, and it may also play a certain role in promoting differentiation of RARα2 positive myeloma cells.

Abstract P6-07-06: Primary prophylaxis of febrile neutropenia during adjuvant docetaxel and cyclophosphamide (TC) chemotherapy for breast cancer

Abstract Background: The combination of docetaxel and cyclophosphamide (TC) for adjuvant treatment of early stage breast cancer improves overall survival compared with doxorubicin and cyclophosphamide (AC) (Jones et al., 2006). Although cardiotoxicity is avoided with TC, the risk of febrile neutropenia (FN) is higher. For TC, reported rates of FN without prophylactic granulocyte colony-stimulating factor (G-CSF) range from 5% in the phase III trial to as high as 46% in retrospective chart reviews. G-CSF is not covered by our provincial cancer funding agency for primary prophylaxis of FN with TC chemotherapy, however it is often prescribed for patients with private insurance. Our aims were twofold: i) to determine the incidence of FN with TC chemotherapy with and without prophylactic G-CSF or antibiotics in two Ontario comprehensive cancer centres, and ii) to evaluate the cost-effectiveness of primary prophylaxis with G-CSF vs. antibiotics. Methods: Patients who received adjuvant TC chemotherapy between January 1, 2008 and December 31, 2012 were identified through pharmacy databases. Electronic charts were retrospectively reviewed to extract patient characteristics, treatment details including G-CSF and antibiotic use, as well as incidence of FN and duration of hospitalization. A Markov model comparing primary G-CSF prophylaxis, primary antibiotic prophylaxis and secondary G-CSF prophylaxis was constructed to compare the cost-effectiveness of these strategies over a four cycle time horizon. Costs were based on resource utilization from this retrospective cohort and supplemented by the published literature, adjusted to 2012 Canadian dollars. The model took the perspective of the third party payer. Both one-way and probabilistic sensitivity analyses were performed. Results: 340 patients were treated with TC over the study period. Of the 73 (21%) who did not receive any primary prophylaxis with G-CSF or antibiotics, 23 (32%) developed FN requiring hospitalization and treatment with intravenous antibiotics. However, only 2 of the 192 patients (1%; P &amp;lt;0.0001) who received primary G-CSF prophylaxis (funded by the patient or a third party payer), and 6 of the 53 patients (11%; P &amp;lt;0.01) who received primary antibiotic prophylaxis (97% receiving ciprofloxacin) developed FN. Age ≥65 was a significant risk factor for FN in the absence of G-CSF (56% vs. 25%, P = 0.02). The results of the cost-effectiveness analysis will be presented at the meeting. Conclusions: The FN rate associated with TC chemotherapy without primary prophylaxis exceeds 30% but may be reduced with prophylactic antibiotics or G-CSF. Unless prophylactic antibiotics are substantially more cost-effective than prophylactic G-CSF for TC chemotherapy in a particular region or country, primary prophylactic G-CSF should be funded, given its greater effectiveness than antibiotics and the global need to minimize the emergence of antibiotic resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-07-06.

G-CSF improves murine G6PC3-deficient neutrophil function by modulating apoptosis and energy homeostasis

AbstractG6PC3 (or glucose-6-phosphatase-β) deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced endoplasmic reticulum (ER) stress, increased apoptosis, impaired energy homeostasis, and impaired functionality. Here we show that murine G6pc3−/− neutrophils undergoing ER stress activate protein kinase-like ER kinase and phosphatidylinositol 3,4,5-trisphosphate/Akt signaling pathways, and that neutrophil apoptosis is mediated in part by the intrinsic mitochondrial pathway. In G6PC3-deficient patients, granulocyte colony-stimulating factor (G-CSF) improves neutropenia, but its impact on neutrophil apoptosis and dysfunction is unknown. We now show that G-CSF delays neutrophil apoptosis in vitro by modulating apoptotic mediators. However, G6pc3−/− neutrophils in culture exhibit accelerated apoptosis compared with wild-type neutrophils both in the presence or absence of G-CSF. Limiting glucose (0.6mM) accelerates apoptosis but is more pronounced for wild-type neutrophils, leading to similar survival profiles for both neutrophil populations. In vivo G-CSF therapy completely corrects neutropenia and normalizes levels of p-Akt, phosphatidylinositol 3,4,5-trisphosphate, and active caspase-3. Neutrophils from in vivo G-CSF–treated G6pc3−/− mice exhibit increased glucose uptake and elevated intracellular levels of G6P, lactate, and adenosine-5′-triphosphate, leading to improved functionality. Together, the results strongly suggest that G-CSF improves G6pc3−/− neutrophil survival by modulating apoptotic mediators and rectifies function by enhancing energy homeostasis.

Phospho- AKT Is Not Sufficient To Delay Neutrophil Apoptosis.

Neutrophils play an important role in the innate immune response and usually are the first cells to defend the organism against infections. The lifetime of neutrophils is tightly regulated and once the mature cells left the bone marrow most of them undergo apoptosis within 48 hours. The rate of neutrophil apoptosis can be modulated by a variety of cytokines, including granulocyte colony-stimulating factor (G-CSF). To investigate the involvement of the G-CSF receptor in the neutrophil susceptibility to apoptosis we utilized mice bearing truncation mutations on the G-CSF receptor (G-CSFR) and also the G:EpoR mutation, where the entire cytoplasmic (signaling) domain of the G-CSFR is replaced with that of the erythropoietin receptor (EpoR). We examined the role of AKT-mTOR pathway in neutrophil apoptosis. Bone marrow neutrophils from wild type (C57BL/6) and mutant mice where cultured in presence or absence of G-CSF and/or Rapamycin (a specific mTor inhibitor); the apoptosis and the level of phosphorylation of AKT and ERK were evaluated. Neutrophils extracted from G:EpoR mice do not respond to G-CSF treatment and undergo apoptosis within 24 hours, although WT neutrophils can be partially protected against apoptosis when treated with G-CSF. Interestingly, G-CSF treatment leads to activation of Akt and Erk kinases, even in G:EpoR neutrophils. Furthermore, the addition of Rapamycin had no effect in the apoptosis of WT neutrophils in presence or absence of G-CSF. To further elucidate the role of the AKT-mTOR pathway, Sca+Lin- WT bone marrow cells were utilized in an in vitro G-CSF dependent granulocytic differentiation assay. The experiments showed that the mTor pathway is important for the early stages of myeloid differentiation but has little influence in the late stages, and also in the life span of neutrophils. Interestingly, over expression of a constitutively active form of AKT (myr-AKT) in Sca+Lin- cells impaired proliferation even though the differentiation was not affected. To overcome this, a tetracycline response element (TRE) conditional expression system was utilized. These experiments showed that myr-AKT expression does not rescue neutrophils from apoptosis. In conclusion, our results indicate that after treatment with G-CSF; AKT and ERK are phosphorylated in WT and G:EpoR neutrophils, despite this, G-CSF does not suppress G:EpoR neutrophil apoptosis while WT neutrophils can have their apoptosis partially delayed. We also found that mTor is involved in the early stages of G-CSF dependent myeloid differentiation and its inhibition can partially arrest differentiation. Conversely, rapamycin does not affect neutrophil apoptosis, in presence or not of G-CSF. In concordance with these results overexpression of a constitutive active form of AKT, myr-AKT does not rescue neutrophils from apoptosis and it is also detrimental to early stages of granulocytic proliferation. These data strongly suggest that the AKT-mTor pathway while important in early myeloid differentiation and proliferation does not play a role in cytokine suppression of neutrophil apoptosis.

Fes Tyrosine Kinase Promotes Survival and Terminal Granulocyte Differentiation of Factor-dependent Myeloid Progenitors (32D) and Activates Lineage-specific Transcription Factors

The c-fps/fes proto-oncogene encodes a 92-kDa protein-tyrosine kinase that is involved in myeloid cell development and function. We have recently shown that expression of an activated allele of Fes (Fes(act)) in monocyte precursors resulted in their differentiation into functional macrophages through the activation of lineage-specific transcription factors. We now report that this kinase also plays a role in the survival and terminal differentiation of granulocyte progenitors. The expression of Fes(act) in factor-dependent 32D cells prevented their apoptotic death after interleukin-3 removal, but Fes(act)-expressing cells remained factor-dependent for proliferation. Removal of interleukin-3 from the Fes(act)-expressing cells was followed by granulocytic differentiation in the absence of granulocyte colony-stimulating factor within 4-8 days. The differentiated cells had distinctive granulocyte morphology and there was up-regulation of CD11b, Gr-1, and late differentiation markers such as lactoferrin, suggesting that this kinase induced terminal granulocytic differentiation. Concomitantly, Fes(act) down-regulated the macrophage marker F4/80, suggesting that the biological activity of Fes was coordinated in a lineage-specific manner. Further analysis showed that Fes(act) caused activation of CCAAT/enhancer-binding protein-alpha and STAT3, two transcription factors that are involved in granulocyte differentiation. Our results provide evidence that Fes may be a key component of the granulocyte differentiation machinery, and suggest a potential mechanism by which this kinase may regulate granulocyte-specific gene expression.

In vitro effect of granulocyte-colony stimulating factor and all-trans retinoic acid on the expression of inflammatory cytokines and adhesion molecules in acute promyelocytic leukemic cells.

Differentiation therapy with all-trans retinoic acid (ATRA) represents a landmark approach in the treatment of acute promyelocytic leukemia (APL). However, a potentially fatal complication of retinoic acid (RA) syndrome occurs in about a quarter of patients and its pathophysiology is still unclear. In order to investigate whether or not the treatment with ATRA leads to increased elaboration of inflammatory cytokines and adhesion molecules by the APL cells, the expression of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-8, L-selectin and intercellular adhesion molecule-1 (ICAM-1) was examined in the APL cells after induction of differentiation with ATRA in the presence or absence of granulocyte-colony stimulating factor (G-CSF) or IL-3 in the present study. Cytokine elaboration by the treated cells was detected using both Northern blotting and enzyme-linked immunosorbent assay. Our results have shown that ATRA induces an increased expression of IL-8, IL-1beta, TNF-alpha and ICAM-1 in APL cells, which can be amplified by the addition of G-CSF. These data imply that the induction of inflammatory cytokines in APL cells may play an important role in the pathogenesis of RA syndrome. Furthermore, G-CSF, through its potent differentiating activity, may increase the risk of such complications during ATRA treatment.

The Role of STAT3 in Granulocyte Colony-stimulating Factor-induced Enhancement of Neutrophilic Differentiation of Me2SO-treated HL-60 Cells: GM-CSF INHIBITS THE NUCLEAR TRANSLOCATION OF TYROSINE-PHOSPHORYLATED STAT3*

The role of granulocyte colony-stimulating factor (G-CSF) on neutrophilic differentiation of Me2SO-treated HL-60 cells was studied. G-CSF augmented the functional maturation of Me2SO-treated HL-60 cells in terms of both O-2-generating ability and expression of the formyl-methionyl-leucyl-phenylalanine receptor. G-CSF induced enhancement of cell growth in Me2SO-treated HL-60 cells. These results indicate that G-CSF is a potent enhancer for the differentiation and proliferation of Me2SO-treated HL-60 cells. G-CSF caused the activation of p70 S6 kinase but not mitogen-activated protein (MAP) kinase. On the other hand, G-CSF rapidly induced tyrosine phosphorylation of signal transducers and activators of transcription-3 (STAT3), but did not induce serine727 phosphorylation. From the analysis of confocal laser scanning fluorescence microscopy and differential centrifugation, it was clearly demonstrated that G-CSF induced nuclear translocation of tyrosine-phosphorylated STAT3. The G-CSF-dependent enhancement of neutrophilic differentiation in Me2SO-HL-60 cells was reversely inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). Notably, in the presence of GM-CSF, G-CSF induced the tyrosine phosphorylation of STAT3 but failed to induce the nuclear translocation of tyrosine-phosphorylated STAT3. GM-CSF induced activation of not only p70 S6 kinase, but also of MAP kinase. Furthermore, GM-CSF caused the rapid serine727 phosphorylation of STAT3, both in the presence and absence of G-CSF. PD98059, an MEK1 inhibitor, inhibited the G-CSF-dependent serine727 phosphorylation of STAT3 and blocked the inhibitory effect of GM-CSF on G-CSF-dependent nuclear translocation of STAT3. These results suggest that G-CSF-dependent nuclear translocation of STAT3 coordinates with the promotion of neutrophilic differentiation in Me2SO-treated HL-60 cells.

Granulocyte Colony-Stimulating Factor Upregulates the Vacuolar Proton ATPase in Human Neutrophils

We have previously shown that granulocyte colony-stimulating factor (G-CSF ) delays spontaneous neutrophil apoptosis through activation of the vacuolar proton ATPase (v-ATPase). We have now examined the regulation of the v-ATPase in neutrophils exposed to G-CSF in vitro. When neutrophils were cultivated in the absence of G-CSF, the 57-kD cytosolic B subunit of the v-ATPase disappeared within 1 to 2 hours, its loss preceding the nuclear changes of apoptosis and coinciding with the onset of acidification. By contrast, in neutrophils cultured for 2 hours in the presence of G-CSF, the amount of the 57-kD subunit was similar to that in freshly isolated neutrophils. However, inhibition of protein synthesis with cycloheximide and actinomycin D led to loss of the 57-kD subunit even in the presence of G-CSF. These results indicated that ongoing protein synthesis was required to maintain the v-ATPase, and further suggested that G-CSF acted, at least in part, by maintaining synthesis of the 57-kD cytosolic subunit. G-CSF also promoted the translocation of the 57-and 33-kD cytosolic v-ATPase subunits to the membrane. Our findings suggested two coordinate mechanisms by which the activity of the v-ATPase could be increased by G-CSF: the synthesis of cytosolic v-ATPase subunits and their translocation to the membrane.

Granulocyte Colony-Stimulating Factor Upregulates the Vacuolar Proton ATPase in Human Neutrophils

AbstractWe have previously shown that granulocyte colony-stimulating factor (G-CSF ) delays spontaneous neutrophil apoptosis through activation of the vacuolar proton ATPase (v-ATPase). We have now examined the regulation of the v-ATPase in neutrophils exposed to G-CSF in vitro. When neutrophils were cultivated in the absence of G-CSF, the 57-kD cytosolic B subunit of the v-ATPase disappeared within 1 to 2 hours, its loss preceding the nuclear changes of apoptosis and coinciding with the onset of acidification. By contrast, in neutrophils cultured for 2 hours in the presence of G-CSF, the amount of the 57-kD subunit was similar to that in freshly isolated neutrophils. However, inhibition of protein synthesis with cycloheximide and actinomycin D led to loss of the 57-kD subunit even in the presence of G-CSF. These results indicated that ongoing protein synthesis was required to maintain the v-ATPase, and further suggested that G-CSF acted, at least in part, by maintaining synthesis of the 57-kD cytosolic subunit. G-CSF also promoted the translocation of the 57-and 33-kD cytosolic v-ATPase subunits to the membrane. Our findings suggested two coordinate mechanisms by which the activity of the v-ATPase could be increased by G-CSF: the synthesis of cytosolic v-ATPase subunits and their translocation to the membrane.

Differential modulation of IL-1-induced endothelial adhesion molecules and transendothelial migration of granulocytes by G-CSF.

Granulocyte colony stimulating factor (G-CSF) is widely used for mobilization of haemopoietic stem cells into the peripheral blood. However, little is known about the mechanisms involved in mobilization and the immune modulatory effects of this growth factor. In this report we show that G-CSF down-regulated intercellular adhesion molecule 1 (ICAM-1) induced by Interleukin-1 (IL-1) on human endothelial cells. Interestingly, the G-CSF-mediated down-modulation of IL-1-induced ICAM-1 appeared to be biphasic. In pharmacological concentrations (> 300 ng/ml), and in dose ranges of plasma G-CSF levels above that of nonfebrile healthy individuals (30 pg/ml), a significant decrease in surface ICAM-1 could be observed. This could be explained, at least in part, by an increased autocrine G-CSF production by endothelial cells in response to IL-1 and exogenous G-CSF. In contrast to ICAM-1, IL-1-triggered VCAM-1 expression was superinduced by G-CSF with the optimal concentration of 30 pg/ml. To evaluate the functional significance of these findings, 51Cr adhesion assays with peripheral blood mononuclear cells (PBMC) or granulocytes known to lack the VCAM-1 counter-receptor very late antigen 4 (VLA-4) and IL-1-stimulated endothelial cells, in the presence or absence of G-CSF, were performed. G-CSF could not inhibit the IL-1-induced adhesion of PBMC to endothelial cells, which may be due to the differential adhesion molecule modulation. In contrast, granulocyte adhesion induced by IL-1 could effectively be blocked by co-incubation with G-CSF. Finally, G-CSF also inhibited transendothelial migration of granulocytes through IL-1-activated endothelial cells in a concentration-dependent manner.

Dimerization of the extracellular domain of granuloycte-colony stimulating factor receptor by ligand binding: a monovalent ligand induces 2:2 complexes.

Granulocyte-colony stimulating factor (G-CSF) binds to a specific cell surface receptor and induces signals for growth and differentiation in cells of granulocyte hematopoietic lineage. In order to understand how G-CSF binding initiates signals into these cells, we have studied its interactions with the entire extracellular domain of the receptor (sG-CSFR). The sG-CSFR was purified from CHO cell conditioned media with a G-CSF affinity column, resting in a preparation fully competent for ligand binding. However, when sG-CSFR was purified by conventional means, i.e., without affinity chromatography, only about half was competent. Therefore, all studies were carried out using affinity-purified material. The sG-CSFR exhibited a weak self-association into a dimer with a dissociation constant of 200microM in the absence of G-CSF. Addition of G-CSF dimerizes the receptor, with a preferred stoichiometry of 2 G-CSF molecules plus 2 receptors. Unexpectedly, receptor-receptor interactions rather than through two receptors binding to the same G-CSF molecule; i.e., G-CSF is a monovalent ligand. G-CSF binding to the receptor monomer occurs with high affinity. The binding of G-CSF also enhances the receptor-receptor dimerization; when G-CSF is bound to both receptors, dimerization is enhanced 2000-fold, while the interaction of a 1:1 receptor-ligand complex with a second ligand-free receptor is enhanced 80-fold. Thus, the mechanism of receptor dimerization is fundamentally different than that of related cytokine receptors such as growth hormone and erythropoietin receptors. Circular dichroic spectra showed a small but significant conformational change of receptor upon binding G-CSF. This is consistent with the idea that G-CSF binding alters the conformation of the receptor, resulting in an increase in receptor-receptor interactions.

Pretreatment of Donor Mice With Granulocyte Colony-Stimulating Factor Polarizes Donor T Lymphocytes Toward Type-2 Cytokine Production and Reduces Severity of Experimental Graft-Versus-Host Disease

The incidence and severity of acute graft-versus-host disease (GVHD) after allogeneic transplantation using peripheral blood progenitor cells mobilized by granulocyte colony-stimulating factor (G-CSF) appear to be no worse than those after bone marrow transplantation, despite the presence of large numbers of T cells in the donor infusion. Experimental studies have shown that type-1 T cells (secreting interleukin-2 [IL-2] and interferon-gamma) mediate acute GVHD, whereas type-2 T cells (secreting IL-4 and IL-10) can prevent acute GVHD. We tested the hypothesis that G-CSF modulates T-cell function toward a type-2 response and thus reduces the severity of acute GVHD. B6 mice were injected with G-CSF or diluent for 4 days, and their splenic T cells were stimulated in vitro with alloantigen or mitogen in the absence of G-CSF. T cells from G-CSF-treated mice showed a significant increase in IL-4 production, with a simultaneous decrease in IL-2 and interferon-gamma production in response to both stimuli. We also examined the effect of G-CSF pretreatment of donors in a GVHD model (B6-->B6D2F1). Survival was significantly improved in recipients of G-CSF-treated donors. Concanavalin-A-induced cytokine production at day 13 after transplantation also showed an increase in IL-4 along with a decrease in IL-2 and IFN-gamma production by splenocytes from recipients of G-CSF-treated bone marrow and T cells. These data show that pretreatment of donors with G-CSF polarizes donor T cells toward the production of type-2 cytokines, which is associated with reduced type-1 cytokine production and reduced severity of acute GVHD.

Inhibition of granulocytic differentiation of acute promyelocytic leukemia cells in primary culture by transforming growth factor-β

We investigated the effect of transforming growth factor- β 1 (TGFβ) on the proliferation and differentiation of cultured acute promyelocytic leukemia (APL) cells with the chromosomal t(15;17) translocation obtained from four patients to determine the role of TGFβ on growth and differentiation of APL cells. DNA synthesis, determined by 3H-thymidine uptake, was inhibited in the presence and absence of granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner by TGFβ in APL cells obtained from three of the four cases. TGFβ and G-CSF did not significantly affect the differentiation of APL cells, but all- trans retinoic acid (RA) induced morphological and functional differentiation in all APL cells tested. G-CSF markedly enhanced RA-induced granulocytic differentiation in APL cells obtained from all four cases. In cells in which TGFβ inhibited DNA synthesis, it also inhibited RA-induced granulocytic differentiation of APL cells and, to a greater degree, granulocytic differentiation induced by RA plus G-CSF. These results suggest that TGFβ is a negative regulator of the proliferation and differentiation of APL cells. The significance of TGFβ as an endogenous regulator in differentiation therapy with RA of APL patients is discussed.