Expression Of Granulocyte Colony-stimulating Factor Research Articles

Map3k8 controls granulocyte colony-stimulating factor production and neutrophil precursor proliferation in lipopolysaccharide-induced emergency granulopoiesis

Map3k8 has been proposed as a useful target for the treatment of inflammatory diseases. We show here that during lipopolysaccharide-induced emergency granulopoiesis, Map3k8 deficiency strongly impairs the increase in circulating mature (Ly6GhighCD11b+) and immature (Ly6GlowCD11b+) neutrophils. After chimaeric bone marrow (BM) transplantation into recipient Map3k8−/− mice, lipopolysaccharide treatment did not increase circulating Ly6GhighCD11b+ cells and strongly decreased circulating Ly6GlowCD11b+ cells. Lipopolysaccharide-treated Map3k8−/− mice showed decreased production of granulocyte colony-stimulating factor (G-CSF), a key factor in neutrophil expansion, and a Map3k8 inhibitor blocked lipopolysaccharide-mediated G-CSF expression in endothelial cell lines. Ly6GlowCD11b+ BM cells from lipopolysaccharide-treated Map3k8−/− mice displayed impaired expression of CCAAT-enhancer-binding protein β, which depends on G-CSF for expression and is crucial for cell cycle acceleration in this life-threatening condition. Accordingly, lipopolysaccharide-treated Map3k8−/− mice showed decreased Ly6GlowCD11b+ BM cell proliferation, as evidenced by a decrease in the percentage of the most immature precursors, which have the highest proliferation capacity among this cell population. Thus, Map3k8 expression by non-haematopoietic tissue is required for lipopolysaccharide-induced emergency granulopoiesis. The novel observation that inhibition of Map3k8 activity decreases neutrophilia during life-threatening systemic infection suggests a possible risk in the proposed use of Map3k8 blockade as an anti-inflammatory therapy.

Potentially probiotic Lactobacillus strains with anti-proliferative activity induce cytokine/chemokine production and neutrophil recruitment in mice.

Lactobacillus pentosus B281 and Lactobacillus plantarum B282 are two Lactobacillus strains previously isolated from fermented table olives. Both strains were found to possess probiotic properties and displayed desirable technological characteristics for application as starters in novel functional food production. In the present study the anti-proliferative and immunostimulatory activities of the two strains were investigated. Firstly, we demonstrated that live L. pentosus B281 and L. plantarum B282 significantly inhibited the growth of human colon cancer cells (Caco-2) in a time- and dose-dependent manner. By employing the air pouch system in mice, we showed that administration of both strains led to a rapid and statistically significant infiltration of leukocytes in the air pouch exudates. The phenotypical characterisation of the recruited immune cells was performed by flow cytometry analysis. We demonstrated that the majority of the infiltrated leukocytes were neutrophils. Finally by using the Mouse Cytokine Array Panel A Detection Antibody cocktail, we showed that both strains induced the expression of granulocyte-colony stimulating factor, interleukin (IL)-1α, IL-1β, IL-6, chemokine (C-X-C motif) ligand (CXCL)-1, chemokine (C-C motif) ligand (CCL)-3, CCL-4, and CXCL-2 and diminished the expression levels of soluble intercellular adhesion molecule, macrophage colony-stimulating factor and metallopeptidase inhibitor 1. Our results showed that both strains display anti-proliferative and immunostimulatory properties equal or even better in some cases than those of established and commonly used probiotic strains. These findings further support the probiotic character of the two strains.

Synonymous codon changes at the 5′-end of the gene strongly impact the heterologous protein expression in Escherichia coli

This study describes the impact of 5′-end codon modulation on the expression of a heterologous gene, human granulocyte colony stimulating factor (GCSF), in Escherichia coli. Fourteen different constructs (pGCSF-01 to pGCSF-14) carrying single or multiple synonymous substitutions at +2, +3 and further down from +4 to +7 codons, were prepared and their expression was monitored in E. coli BL21 Codon-Plus (DE3) RIPL using a strong T7 lac-promoter based expression system. A single nucleotide change at +2 Thr codon (ACC→ACA) either alone or in combination with +3 Pro codon (CCC/CCT/CCA) resulted in the expression enhancement of an otherwise poorly expressed native-GCSF, to a level that corresponded to 45–50% of the total E. coli BL21 CodonPlus (DE3) RIPL cellular proteins. The differences in GCSF expression amongst different constructs could be attributed to the preferential or non-preferential codon usage, reduced number of G/C nucleotides and the stability of mRNA secondary structure formed near the 5′-end coding region. The expression of GCSF achieved was in the form of biologically inactive inclusion bodies that were solubilized using mild concentration of a non-ionic surfactant and refolded by a simplified, step-dialysis approach. Biological activity of the purified GCSF, assessed in induced neutropenic mice, was similar to the commercially available preparation of the GCSF analog (filgrastim).

Enhancing granulocyte colony-stimulating factor expression in Pichia pastoris through fusion with human serum albumin

ABSTRACTProtein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in Pichia pastoris by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184 bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of α-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZαB. Excreted protein in the range of 110–380 mg L−1 was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.

Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro

BackgroundInflammation is a key contributor to central nervous system (CNS) injury such as stroke, and is a major target for therapeutic intervention. Effective treatments for CNS injuries are limited and applicable to only a minority of patients. Stem cell-based therapies are increasingly considered for the treatment of CNS disease, because they can be used as in-situ regulators of inflammation, and improve tissue repair and recovery. One promising option is the use of bone marrow-derived mesenchymal stem cells (MSCs), which can secrete anti-inflammatory and trophic factors, can migrate towards inflamed and injured sites or can be implanted locally. Here we tested the hypothesis that pre-treatment with inflammatory cytokines can prime MSCs towards an anti-inflammatory and pro-trophic phenotype in vitro.MethodsHuman MSCs from three different donors were cultured in vitro and treated with inflammatory mediators as follows: interleukin (IL)-1α, IL-1β, tumour necrosis factor alpha (TNF-α) or interferon-γ. After 24 h of treatment, cell supernatants were analysed by ELISA for expression of granulocyte-colony stimulating factor (G-CSF), IL-10, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), IL-1 receptor antagonist (IL-1Ra) and vascular endothelial growth factor (VEGF). To confirm the anti-inflammatory potential of MSCs, immortalised mouse microglial BV2 cells were treated with bacterial lipopolysaccharide (LPS) and exposed to conditioned media (CM) of naïve or IL-1-primed MSCs, and levels of secreted microglial-derived inflammatory mediators including TNF-α, IL-10, G-CSF and IL-6 were measured by ELISA.ResultsUnstimulated MSCs constitutively expressed anti-inflammatory cytokines and trophic factors (IL-10, VEGF, BDNF, G-CSF, NGF and IL-1Ra). MSCs primed with IL-1α or IL-1β showed increased secretion of G-CSF, which was blocked by IL-1Ra. Furthermore, LPS-treated BV2 cells secreted less inflammatory and apoptotic markers, and showed increased secretion of the anti-inflammatory IL-10 in response to treatment with CM of IL-1-primed MSCs compared with CM of unprimed MSCs.ConclusionsOur results demonstrate that priming MSCs with IL-1 increases expression of trophic factor G-CSF through an IL-1 receptor type 1 (IL-1R1) mechanism, and induces a reduction in the secretion of inflammatory mediators in LPS-activated microglial cells. The results therefore support the potential use of preconditioning treatments of stem cells in future therapies.

Expression of granulocyte colony-stimulating factor 3 receptor in the spinal dorsal horn following spinal nerve ligation-induced neuropathic pain

In previous studies that have profiled gene expression in patients with complex regional pain syndrome (CRPS), the expression of granulocyte colony-stimulating factor 3 receptor (G-CSFR) was elevated, as were a number of pain-associated genes. The present study determined the expression of G-CSFR and the mechanisms by which it may affect hypersensitivity, focusing on the signal transducer and activator of transcription 3 (STAT3)/transient receptor potential cation channel subfamily V 1 (TRPV1) signaling pathway in particular, which is an important mediator of pain. Following L5 spinal nerve ligation (SNL) surgery, the protein and mRNA levels of G-CSFR increased in the ipsilateral spinal dorsal horn when compared with the sham and/or contralateral control. Double immunofluorescence further demonstrated that G-CSFR colocalized with TRPV1 and phosphorylated STAT in the neurons of the spinal dorsal horn. G-CSF treatment led to an increase in G-CSFR and TRPV1 expression and phosphorylation of STAT3. These results indicate that G-CSF-induced G-CSFR expression may activate TRPV1 by promoting phosphorylation of STAT3. Collectively, the results suggest, for the first time, that the expression of G-CSFR in neurons following peripheral nerve injury may be involved in the induction and maintenance of neuropathic pain through the STAT3 and TRPV1 signaling pathway.

顆粒球コロニー刺激因子の発現を認めた限局性悪性胸膜中皮腫の1切除例

顆粒球コロニー刺激因子の発現を認めた限局性悪性胸膜中皮腫の1切除例

Altered Expression of Hematopoiesis Regulatory Genes in the Bone Marrow Mesenchymal Stem Cells of Patients of Aplastic Anemia

Mesenchymal stem cells (MSC) are an important component of the hematopoietic niche in the bone marrow (BM) and regulate hematopoiesis by producing a variety of cytokines and growth factors. In aplastic anemia (AA), most of the studies have attributed the reduced hematopoiesis to a defect in hematopoietic stem cells (HSC) and limited data is available on the role of BM-MSC in AA. Therefore, the objective of the present study was to evaluate the expression of hematopoiesis regulatory genes, viz. granulocyte colony stimulating factor (G-CSF), stromal cell derived factor (SDF-1), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-α) macrophage inflammatory protein-1 alpha (MIP-1α) and transforming growth factor-beta (TGF-β) in BM-MSC of patients with AA and compare it with BM-MSC of control group.Twenty patients of idiopathic acquired AA with a median age of 25.5 years (range: 12-64 years) were included in the study. The control group consisted of 10 healthy volunteers and 10 patients with iron deficiency anemia or immune thrombocytopenic purpura. The median age of the control group was 20 years (range: 11-62 years). The BM-MSC were isolated and cultured as per protocol standardized and previously published by us. Third passage cells were used in the study. The MSC were characterized both by their phenotypic markers and by their ability to differentiate into adipogenic and osteogenic lineages. The expression of hematopoiesis regulatory genes was studied by real-time quantitative polymerase chain reaction (qRT-PCR). The GAPDH was used as the housekeeping gene to normalize the transcript levels and the fold change in the gene expression was calculated by 2-ΔΔCtmethod.The BM-MSC of AA patients and controls had similar morphology and expression of mesenchymal markers CD73, CD105, CD90 and CD166, absence of expression of hematopoietic markers CD13, CD34 and CD45 and of HLA-DR. However, the BM-MSC of AA patients exhibited a higher adipogenic and a lower osteogenic differentiation in comparison to those of controls. Further, the BM-MSC of AA patients in comparison to those of control group, had a higher expression of G-CSF (fold increase: 1.99; p<0.0001), SDF-1 (fold increase: 1.37; p<0.01) and TNF-α (fold increase: 10.68; p<0.0001) and a very low expression of MIP-1α (fold decease: 50.0; p<0.0001) transcripts. The expression of SCF and TGF-β transcripts were comparable in the BM-MSC of both the groups (p>0.05).Though AA patients have been shown to have elevated levels of G-CSF in the peripheral blood and BM but there is only one previous report on G-CSF gene expression in BM-MSC of AA, in which a higher expression was observed and thus corroborates with our data. There is no data available on SDF-1 levels in the peripheral blood and bone marrow of AA patients. We have observed higher gene expression of SDF-1 in BM-MSC of AA patients. The higher expression of G-CSF and SDF-1, pro-hematopoietic factors, in AA may be due to a compensatory response of the BM stroma to boost the hematopoiesis. Our observation of higher TNF-α gene expression in BM-MSC corroborates with previous reports on higher levels of this anti-hematopoietic cytokine in the BM plasma of patients with AA and indicates that MSC could contribute to the increase in the TNF-α level in the BM of AA patients. A conspicuous observation of our study was a markedly decreased expression of MIP-1α gene in BM-MSC of AA and to the best of our knowledge this is the first report on MIP-1α in AA. MIP-1α is a chemokine which has been shown to inhibit proliferation of HSC in vitro and thus may help to maintain HSC in an undifferentiated state. Furthermore, MIP-1α has also been reported to mediate interaction of HSC with stromal cells in BM and may have a role in supporting hematopoiesis. Its precise role in AA needs to be studied further. We are currently studying the levels of these cytokines/growth factors in the BM plasma of the same cohort of AA patients and controls and the data will be presented.Our study thus shows that BM-MSC of AA patients have altered expression of hematopoiesis regulatory genes which may contribute to the pathobiology of the disease. DisclosuresNityanand:Sanjay Gandhi Post Graduate Institute of Medical Sciences: Employment, Research Funding. Tripathy:Sanjay Gandhi Post Graduate Institute of Medical Sciences: Employment. Chaturvedi:Dept of Biotechnology, Govt of India: Employment. Minocha:Dept of Science and Technology, Govt of India: Other: PhD scholarship. Sharma:Sanjay Gandhi Post Graduate Institute of Medical Sciences: Employment. Rahman:SGPGI, Lucknow , India: Employment, Research Funding.

Honeybee product therapeutic as stem cells homing for ovary failure.

Aim:Complexity of the method of isolation, cultivation in vitro and the expensive cost of transplantation process of stem cells, it would require an innovation to homing and differentiation of stem cells and increase folliculogenesis. The stem cells homing was achieved through the provision of food or beverages derived from natural materials like honeybee product. Through honeybee product, there will be homing of stem cells and accompany with the sources from the body itself will take place in regeneration of the ovary.Materials and Methods:Female rats model of degenerative ovary was obtained through food fasting but still have drinking water for 5 days. It caused malnutrition and damage of the ovarian tissue. The administration of 50% honeybee product (T1) was performed for 10 consecutive days, while the positive control group (T0+) was fasted and not given honeybee product and the negative control (T0−) not fasted and without honeybee product. Observations were taken for homing of stem cells, raised of folliculogenesis, differentiation of stem cells, and regeneration of the ovarian tissue using routine H&E staining.Results:Homing of stem cells shown the vascular endothelial growth factor and granulocyte colony-stimulating factor expression; enhancement of folliculogenesis was indicated by an increase of follicle dee Graaf count; enhancement of differentiation of stem cells was indicated by growth differentiation factor-9 expression; and regeneration of ovarian tissue indicated by intact ovarian tissue with growing follicles.Conclusion:Honeybee product can be induced endogenous stem cells in regeneration of ovary failure due to malnutrition.

Characterization of recombinant human granulocyte colony-stimulating factor expression by FT-IR spectroscopy: Studies on thermal induction and media formulation on the stability of the protein secondary structure

ABSTRACTThe Fourier-transform infrared (FT-IR) spectroscopic approach has been employed to understand the recombinant human G-CSF (rhG-CSF) protein accumulation, secondary structure, and thermal stability in Escherichia coli grown under a temperature shift strategy (37 and 28°C) in various media formulations. The choline + sodium pyruvate (37°C) and sodium pyruvate (28°C) formulations have shown the highest inclusion body (IB) accumulation of 0.41 and 0.46 mg/mL, respectively. Furthermore, insights on the structure of the rhG-CSF within IBs and intact cells have been investigated through secondary structure analysis. Thermal stability experiments were also carried out to explain the pattern of the second derivative structure of rhG-CSF. The studies showed that choline + sodium pyruvate formulation has preserved the protein secondary structure even at 82°C. Overall, the FT-IR spectroscopic technique can also be adopted to accelerate the characterization of other recombinant therapeutic proteins of E. coli origin.

Keratinocyte-Releasable Factors Stimulate the Expression of Granulocyte Colony-Stimulating Factor in Human Dermal Fibroblasts.

Interaction between keratinocytes and fibroblasts plays a critical role in maintaining skin integrity under both normal and pathological conditions. We have previously demonstrated that keratinocyte-releasable factors influence the expression of key extracellular matrix components, such as collagen and matrix metalloproteinases in dermal fibroblasts. In this study, we utilized DNA microarray analysis to examine the effects of keratinocyte-releasable factors on the expression of several cytokines in human dermal fibroblasts. The results revealed significantly higher granulocyte colony-stimulating factor (G-CSF) expression in fibroblasts co-cultured with keratinocytes relative to mono-cultured cells, which was verified by RT-PCR and western blot. G-CSF is an important hematopoietic factor also thought to play a beneficial role in wound healing through stimulating keratinocyte proliferation. To partially characterize the keratinocyte-releasable factors responsible for stimulating G-CSF production, keratinocyte-conditioned medium (KCM) was subjected to thermal treatment and ammonium sulfate precipitation before treating fibroblasts. The results showed that keratinocyte-releasable G-CSF-stimulating factors remain stable at 56°C and upon 50% ammonium sulfate precipitation. Knowing that keratinocytes release IL-1, which stimulates G-CSF expression in various immune cells, several experiments were conducted to ask whether this might also be the case for fibroblasts. The results showed that the addition of recombinant IL-1 markedly increased G-CSF expression in fibroblasts; however, IL-1 receptor antagonist only partially abrogated KCM-stimulated G-CSF expression, indicating the role of additional keratinocyte-releasable factors. These findings underline the importance of cross-talk between keratinocytes and fibroblasts, suggesting that communication between these cells in vivo modulates the production of cytokines required for cutaneous wound healing and maintenance. J. Cell. Biochem. 118: 308-317, 2017. © 2016 Wiley Periodicals, Inc.

TLR9 expression is required for the development of cigarette smoke-induced emphysema in mice.

The expression of Toll-like receptor (TLR)-9, a pathogen recognition receptor that recognizes unmethylated CpG sequences in microbial DNA molecules, is linked to the pathogenesis of several lung diseases. TLR9 expression and signaling was investigated in animal and cell models of chronic obstructive pulmonary disease (COPD). We observed enhanced TLR9 expression in mouse lungs following exposure to cigarette smoke. Tlr9(-/-) mice were resistant to cigarette smoke-induced loss of lung function as determined by mean linear intercept, total lung capacity, lung compliance, and tissue elastance analysis. Tlr9 expression also regulated smoke-mediated immune cell recruitment to the lung; apoptosis; expression of granulocyte-colony stimulating factor (G-CSF), the CXCL5 protein, and matrix metalloproteinase-2 (MMP-2); and protein tyrosine phosphatase 1B (PTP1B) activity in the lung. PTP1B, a phosphatase with anti-inflammatory abilities, was identified as binding to TLR9. In vivo delivery of a TLR9 agonist enhanced TLR9 binding to PTP1B, which inactivated PTP1B. Ptp1b(-/-) mice had elevated lung concentrations of G-CSF, CXCL5, and MMP-2, and tissue expression of type-1 interferon following TLR9 agonist administration, compared with wild-type mice. TLR9 responses were further determined in fully differentiated normal human bronchial epithelial (NHBE) cells isolated from nonsmoker, smoker, and COPD donors, and then cultured at air liquid interface. NHBE cells from smokers and patients with COPD expressed more TLR9 and secreted greater levels of G-CSF, IL-6, CXCL5, IL-1β, and MMP-2 upon TLR9 ligand stimulation compared with cells from nonsmoker donors. Although TLR9 combats infection, our results indicate that TLR9 induction can affect lung function by inactivating PTP1B and upregulating expression of proinflammatory cytokines.

Effect of icariin on the proteomic expression profile of bone microvascular endothelial cells of human femoral head against steroids-induced lesion

To explore the effect of icariin on the proteomic expression profile of bone microvascular endothelial cells of human femoral head against steroids-induced lesion using protein chip system in vitro. Bone microvascular endothelial cells (BMECs) in cancellous bone of femoral head were isolated and harvested in vitro.The model of BMECs injury induced by glucocorticoid and icariin preconditioning was established. The apoptosis of BMECs were detected with terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)methods. The proteomic expression profile of BMECs in each group were detected by protein chip system. The apoptosis rates of hydrocortisone groups (46.9%±5.8%, 78.0%±8.5%) were significantly higher than that of normal group(4.8%±1.3%), meanwhile the apoptosis rate of icariin+ hydrocortisone groups(15.2%±7.7%, 44.6%±5.3%)significantly decreased compared with hydrocortisone group (P<0.001). The results of protein chip showed that the expression of granulocyte colony-stimulating factor (G-CSF) in the hydrocortisone group decreased compared with the control group, meanwhile the expression of IL-4 increased. The expression of G-CSF in the icariin+ hydrocortisone groups increased compared with the hydrocortisone group, meanwhile the expression of IL-4 decreased.No appreciable change was found in the expression of apoptotic factors. The therapy effect of icariin for steroid-induced necrosis of femoral head might be related with the protective action to BMECs and the moderating effect of promoting the proteins expression of pro-angiogenesis factor and inhibiting expression of inflammatory factor.

Expression of G-CSF and clinical pathological significance in cervical cancer

To observe the expression of granulocyte colony-stimulating factor (G-CSF) in cervical cancer and to investigate its clinical significance. A total of 53 cases of cervical cancer with a large number of granular cell infiltration were collected in the last ten years, and 57 cases of cervical cancer with no cell infiltration were selected as controls. The infiltration of neutrophils in cancer tissues was observed, and the expression of G-CSF in cancer tissue was detected by immunohistochemistry at the same time. And the relationship between the expression of G-CSF and clinical pathological significance in cervical cancer was analyzed. All the patients were followed up, and the effect of G-CSF on the prognosis of the patients was analyzed. Expression of G-CSF was observed in 53 cases out of the 110 cases cervical cancer. Among them, 48 (90.6%) cases were squamous cell carcinoma, 5(9.4%) cases were adenocarcinoma. The expression of G-CSF was closely related to granulocyte infiltration, histological type, necrosis, high grading and regional lymph node metastasis (P<0.01), but not related to size of tumor (P>0.05). G-CSF positive cases were 33.676 and 78.495 times more dangerous than those cases that were negative in high grading and regional lymph node metastasis (P<0.01). The difference in survival between the positive and the negative was also significant (P<0.01). Certain cervical cancer can produce G-CSF, and the most common in non-keratinizing squamous cell carcinoma. Cervical cancer tissue that excretes G-CSF tends to have low differentiation, obvious dysplasia, high degree of malignancy, extensive necrosis and granulocyte cell infiltration, and is often associated with lymph node metastasis, with low survival rate and poor prognosis.

SB203580 increases G-CSF production via a stem-loop destabilizing element in the 3' untranslated region in macrophages independently of its effect on p38 MAPK activity.

BackgroundGranulocyte-colony stimulating factor (G-CSF) is a major regulator of the production and survival of neutrophils. Regulation of G-CSF expression is complex and occurs at both transcription and post-transcription levels. Two distinct types of cis-acting elements in the 3’ untranslated region (3’UTR) of G-CSF mRNA have been identified as destabilizing elements; these consist of adenylate uridylate-rich elements (AUREs) and a stem–loop destabilizing element (SLDE). Regulation of the stability of mRNA by p38 mitogen-activated protein kinase (MAPK) has been indicated to be linked to AUREs in the 3’UTR. However, whether p38 MAPK is involved in the regulation of the stability of G-CSF mRNA has not been elucidated. This study investigated the effect of SB203580, an inhibitor of p38 MAPK, on the lipopolysaccharide-induced G-CSF expression in macrophages at the post-transcription level.ResultsOur study showed surprising results that SB203580 augmented the lipopolysaccharide-induced increase in the G-CSF mRNA levels in RAW264.7 mouse macrophages, mouse bone marrow-derived macrophages and in THP-1 human macrophages. This effect was also seen in p38α MAPK knockdown RAW264.7 cells, showing that it was not due to inhibition of p38 MAPK activity. In the presence of actinomycin D, the decay of G-CSF mRNA was slower in SB203580-treated cells than in control cells, showing that SB203580 increased the stability of G-CSF mRNA. Reporter genes containing luciferase with or without the 3’UTR of G-CSF were constructed and transfected into RAW264.7 cells and the results showed that the presence of the 3’UTR reduced the luciferase mRNA levels and luciferase activity. Furthermore, SB203580 increased the luciferase mRNA levels and activity in RAW264.7 cells transfected with the luciferase reporter containing the 3’UTR, but not in cells transfected with the luciferase reporter without the 3’UTR. Mutations of the highly conserved SLDE in the 3’UTR abolished these effects, showing that the SLDE was essential for the SB203580-induced increase in the stability of mRNA.ConclusionsSB203580 increases G-CSF expression in macrophages by increasing the stability of G-CSF mRNA via its 3’UTR, and the effect was not due to its inhibition of p38 MAPK activity. The results of this study also highlight a potential target for boosting endogenous production of G-CSF during neutropenia.

The significance of G-CSF expression and myeloid-derived suppressor cells in the chemoresistance of uterine cervical cancer.

Granulocyte-colony stimulating factor (G-CSF) producing malignant tumor has been reported to occur in various organs, and has been associated with poor clinical outcome. The aim of this study is to investigate the significance of tumor G-CSF expression in the chemosensitivity of uterine cervical cancer. The clinical data of recurrent or advanced cervical cancer patients who were treated with platinum-based chemotherapy were analyzed. Clinical samples, cervical cancer cell lines, and a mouse model of cervical cancer were employed to examine the mechanisms responsible for the development of chemoresistance in G-CSF-producing cervical cancer, focusing on myeloid-derived suppressor cells (MDSC). As a result, the tumor G-CSF expression was significantly associated with increased MDSC frequencies and compromised survival. In vitro and in vivo experiments demonstrated that the increased MDSC induced by tumor-derived G-CSF is involved in the development of chemoresistance. The depletion of MDSC via splenectomy or the administration of anti-Gr-1 antibody sensitized G-CSF-producing cervical cancer to cisplatin. In conclusion, tumor G-CSF expression is an indicator of an extremely poor prognosis in cervical cancer patients that are treated with chemotherapy. Combining MDSC-targeting treatments with current standard chemotherapies might have therapeutic efficacy as a treatment for G-CSF-producing cervical cancer.

G-CSF regulates macrophage phenotype and associates with poor overall survival in human triple-negative breast cancer

ABSTRACTTumor-associated macrophages (TAMs) have been implicated in the promotion of breast cancer growth and metastasis, and a strong infiltration by TAMs has been associated with estrogen receptor (ER)-negative tumors and poor prognosis. However, the molecular mechanisms behind these observations are unclear. We investigated macrophage activation in response to co-culture with several breast cancer cell lines (T47D, MCF-7, BT-474, SKBR-3, Cal-51 and MDA-MB-231) and found that high granulocyte colony-stimulating factor (G-CSF) secretion by the triple-negative breast cancer (TNBC) cell line MDA-MB-231 gave rise to immunosuppressive HLA-DRlo macrophages that promoted migration of breast cancer cells via secretion of TGF-α. In human breast cancer samples (n = 548), G-CSF was highly expressed in TNBC (p < 0.001) and associated with CD163+ macrophages (p < 0.0001), poorer overall survival (OS) (p = 0.021) and significantly increased numbers of TGF-α+ cells. While G-CSF blockade in the 4T1 mammary tumor model promoted maturation of MHCIIhi blood monocytes and TAMs and significantly reduced lung metastasis, anti-CSF-1R treatment promoted MHCIIloF4/80hiMRhi anti-inflammatory TAMs and enhanced lung metastasis in the presence of high G-CSF levels. Combined anti-G-CSF and anti-CSF-1R therapy significantly increased lymph node metastases, possibly via depletion of the so-called “gate-keeper” subcapsular sinus macrophages. These results indicate that G-CSF promotes the anti-inflammatory phenotype of tumor-induced macrophages when CSF-1R is inhibited and therefore caution against the use of M-CSF/CSF-1R targeting agents in tumors with high G-CSF expression.

Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris.

Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.

Abstract 1416: Granulocyte-colony stimulating factor (G-CSF) enhances stemness by inducing myeloid-derived suppressor cell (MDSC) in cervical cancer

Abstract Objectives: Granulocyte-colony stimulating factor (G-CSF) is known to induce the myeloid-derived suppressor cell (MDSC) to stimulate the progression of uterine cervical cancer. The aim of the current study is to investigate the role of MDSC in the induction of cancer stem cells in uterine cervical cancer. Methods: We first established cervical cancer cell lines stably transfected with G-CSF expression vector (ME180-GCSF) or control vector (ME180-control). Then, using BALB/c nude mice subcutaneously inoculated with ME180-G-CSF or ME180-control cells, the effect of G-CSF on the induction of MDSCs (CD11b+Gr1+ cells) and cancer stem cells (ALDH-high cells) were assessed by flow cytometry. Using MDSCs isolated from the spleens of ME180-GCSF-bearing mice, we next investigated whether the MDSCs induce cancer stem cells in vitro. Finally, we analyzed the association between the G-CSF expression and ALDH1A1 expression in human cervical cancer specimens by immunohistochemistry. Results: 1) G-CSF induces MDSCs from mice bone marrow. 2) ME180-GCSF-derived tumors contain greater number of MDSCs and ALDH-high cells. 3) Co-incubation of ME180 with MDSCs cells increased the number of ALDH-high cells. 4) A positive correlation was observed between the tumor G-CSF expression and ALDH1A1 expression in human cervical cancers. Conclusions: G-CSF-induced MDSCs increase the number of cancer stem cells in uterine cervical cancer. Citation Format: Hiromasa Kuroda. Granulocyte-colony stimulating factor (G-CSF) enhances stemness by inducing myeloid-derived suppressor cell (MDSC) in cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1416. doi:10.1158/1538-7445.AM2015-1416

Abstract 438: Granulocyte-colony stimulating factor (G-CSF) facilitates metastasis of uterine cervical cancer by recruiting myeloid-derived suppressor cells (MDSCs) via CXCL12-CXCR4 axis

Abstract Objectives: Granulocyte-colony stimulating factor (G-CSF) is known to induce the myeloid-derived suppressor cells (MDSCs) to stimulate the progression of uterine cervical cancer. The aim of this study was to investigate the role of G-CSF and the MDSCs in the invasion and the metastasis of uterine cervical cancer. Methods: We first established cervical cancer cell lines stably transfected with G-CSF expression vector (ME180-GCSF) or control vector (ME180-control). Then, using mice subcutaneously inoculated with ME180-control or ME180-GCSF cells, the effect of G-CSF on the induction of MDSCs (CD11b+Gr1+ cells) and the metastatic activity of cervical cancer cells. The association of MDSCs infiltration and the clinical outcome was also assessed immunohistochemically using tumor samples obtained from cervical cancer patients. Finally, using MDSCs isolated from the spleen of the ME180-GCSF-bearing mice, we investigated whether the MDSCs facilitate the invasive or metastatic activities of uterine cervical cancer in vitro and in vivo. Results: 1) ME180-GCSF-derived tumors contain greater number of MDSC, more frequently develop lymphovascular space invasion, and developed greater number of metastatic tumors than ME180-control-tumors. 2) Immunohistochemical analyses using human samples revealed that increased MDSC counts were significantly associated with deep stromal invasion and lymphovascular space invasion. 3) MDSCs were recruited into cervical cancer via CXCL12-CXCR4 axis. 4) Coincubation of MDSCs with ME180 cells increased the invasive activity of ME180 cells. 5) The depletion of MDSCs significantly decreased the invasive and metastatic activities of ME180 cells. Conclusions: Granulocyte-colony stimulating factor (G-CSF) facilitates metastasis by recruiting MDSCs. MDSCs and CXCL12-CXCR4 axis might be promising therapeutic targets in patients with uterine cervical cancer. Citation Format: Tomoyuki Sasano, Seiji Mabuchi, Ryoko Takahashi, Hiromasa Kuroda, Kenjiro Sawada, Tadashi Kimura. Granulocyte-colony stimulating factor (G-CSF) facilitates metastasis of uterine cervical cancer by recruiting myeloid-derived suppressor cells (MDSCs) via CXCL12-CXCR4 axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 438. doi:10.1158/1538-7445.AM2015-438