Truncated RUNX1 Generated by the Fusion of RUNX1 to Antisense GRIK2 via a Cryptic Chromosome Translocation Enhances Sensitivity to Granulocyte Colony-Stimulating Factor.

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.

Immunohistochemical Study on the Expression of G-CSF, G-CSFR, VEGF, VEGFR-1, Foxp3 in First Trimester Trophoblast of Recurrent Pregnancy Loss in Pregnancies Treated with G-CSF and Controls.

BACKGROUND: Recurrent Pregnancy Loss (RPL) is a syndrome recognizing several causes, and in some cases the treatment with Granulocyte Colony Stimulating Factor (G-CSF) may be successful, especially when karyotype of the previous miscarriage showed no embryo chromosomal abnormalities. In order to evaluate the effects of G-CSF treatment on the decidual and trophoblast expression of G-CSF and its receptor, VEGF and its receptor and Foxp3, specific marker of putative Tregs we conducted an immunohistochemical study. METHODS: This study was conducted on three groups of patients for a total of 38 women: in 8 cases decidual and trophoblast tissue were obtained from 8 women with unexplained RPL treated with G-CSF that miscarried despite treatment; in 15 cases the tissue were obtained from 15 women with unexplained RPL no treated; 15 cases of women who underwent voluntary pregnancy termination were used as controls. Tissue collected from these patients were used for immunohistochemistry studies testing the expression of G-CSF, G-CSFR, VEGF, VEGFR-1 and Foxp3. RESULTS: G-CSF treatment increased the concentration of cells expressing Foxp3, specific marker for Tregs, in the decidua, whereas in no treated RPL a reduction of these cells was found when compared to controls. Furthermore, G-CSF treatment increased the expression of G-CSF and VEGF in the trophoblast. CONCLUSIONS: Our study showed that G-CSF treatment increased the number of decidual Treg cells in RPL patients as well as the expression of G-CSF and VEGF in villus trophoblast. These finding may explain the effectiveness of this treatment in RPL, probably regulating the maternal immune response through Tregs recruitment in the decidua, as well as stimulating trophoblast growth.

Vitamin Е activates expression of С/EBP alpha transcription factor and G-CSF receptor in leukemic K562 cells.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the activity of BCR-ABL fusion oncogene. Tyrosine kinase inhibitors are the current treatment of CML, but secondary mutations finally contribute to therapy resistance and blast crisis of the disease. The search for the novel compounds for the effective control of CML is now in the spotlight. The progression of CML to blast crisis is correlated with down-modulation of C/EBP alpha. Therefore, C/EBP alpha may be considered as a putative target in differentiation therapies in myeloid leukemias. The aim of the study was to assess the potential of vitamin E as the possible inducer of C/EBP alpha expression in BCR-ABL-positive CML K562 cells. MATERIALS AND METHODS: RNA extracted from K562 cells cultured with valproic acid or vitamin E was converted to cDNA, RT-PCR reactions were carried out using HotStarTaq DNA polymerase with primers for C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR). RESULTS: We have not found detectable expression of C/EBP alpha in K562 cells. Upon 48-h culture with vitamin E at a dose of 100 µM, K562 cells expressed both C/EBP alpha and G-CSFR. CONCLUSION: Vitamin E restored the expression of C/EBP alpha mRNA in chronic myelogenous leukemia K562 cells. In this setting, G-CSFR expression in vitamin E treated K562 cells seems to suggest the activation to granulocytic differentiation. It should be further elucidated whether such effects of vitamin E on C/EBP alpha transcription factor are direct or mediated indirectly due to antioxidant properties of vitamin E.

Expression and role of granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) and granulocyte colony-stimulating factor receptor (G-CSFR) on Ph-positive acute B lymphoblastic leukemia.

OBJECTIVE: We observed that ph + ALL patients administrated with recombinant human G-CSF (rhG-CSF) after intense chemotherapy have presented a trend of disease relapse. Thus, we aim to thoroughly investigate the expression and role of GM-CSFR and G-CSFR on ph + ALL patients. METHOD: SUP-B15, BALL-1 and primary leukemia cells were used in this study. Transcript levels were analyzed by quantitative PCR while cell viability was measured using a CCK-8 assay. Flow cytometry was used to assess the different stages of cell cycle. RESULTS: We found that the mRNA expression levels of GM-CSFR and G-CSFR were higher in patients with ph + ALL, as well as in SUP-B15 cells. rhG-CSF was also observed to promote the viability of SUP-B15 cells while inversely inhibiting BALL-1 cell viability. In addition, we also determined that rhG-CSF (100 ng/ml) decreased the sensitivity of SUP-B15 cells to imatinib and nilotinib, while the results were exactly the contrary for dasatinib. CONCLUSION: We demonstrated high expression levels of GM-CSFR and G-CSFR, as well as their promotable role for viability in ph + ALL cells. We further found that rhG-CSF influenced the sensitivity of SUP-B15 cells to TKIs.

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017.

CONCENTRATIONS OF VITREAL CYTOKINES IN RHEGMATOGENOUS RETINAL DETACHMENT.

UNLABELLED: Proliferative vitreoretinopathy (PVR) is one of the most frequent causes of failure of rhegmatogenous retinal detachment (RRD) surgery. AIM: To measure the vitreous levels of granulocyte colony stimulating factor (G-CSF) and monocyte chemoattractant protein 1 (MCP-1) in eyes with RRD and in a control group. MATERIAL AND METHODS: A prospective study of 40 patients operated for RRD (study group) and 20 patients with epiretinal membrane or macular holes (selected as control group since they needed vitrectomy but had attached retinas). Vitreous samples were collected during vitrectomy and were assessed for the presence of cytokines using a fluorescent bead-based multiplex assay. RESULTS: The concentration of G-CSF (8.59 pg/ml) and MCP-1 (1615.2 pg/ml) were significantly increased in the study group, when compared to the control group (0 and 469.13 pg/ml, respectively). MCP-1 was also significantly increased in the subgroup of patients with PVR compared to the patients with uncomplicated RRD. CONCLUSIONS: The levels of these biomarkers support the idea that proliferative vitreoretinopathy has an inflammatory component.

A soluble granulocyte colony stimulating factor decoy receptor as a novel tool to increase hematopoietic cell homing and reconstitution in mice.

The relative ineffectiveness of hematopoietic stem cells in reaching the bone marrow upon transplantation combined with the limited number of these cells available is a major reason for graft failure and delayed hematopoietic recovery. Hence, the development of strategies that could enhance homing is of high interest. Here, we provide evidence that homing is severely impaired postexposure to ionizing radiation (IR) in mice, an effect we found was time dependent and could be partially rescued using mesenchymal stromal cell (MSC) therapy. In an attempt to further increase homing, we took advantage of our observation that the granulocyte colony stimulating factor (G-CSF), a cytokine known to induce cell mobilization, is increased in the marrow of mice shortly after their exposure to IR. As such, we developed a truncated, yet functional, soluble G-CSF receptor (solG-CSFR), which we hypothesized could act as a decoy and foster homing. Using MSCs or conditioned media as delivery vehicles, we show that an engineered solG-CSFR has the potential to increase homing and hematopoietic reconstitution in mice. Altogether, our results provide novel findings at the interplay of IR and stromal cell therapy and present the regulation of endogenous G-CSF as an innovative proof-of-concept strategy to manipulate hematopoietic cell homing.

Granulocyte-colony-stimulatory factor: a strong inhibitor of natural killer cell function.

BACKGROUND: The human cytokine granulocyte-colony stimulatory factor (G-CSF) has found widespread application in the medical treatment of neutropenia and to mobilize hematopoietic stem cells used for transplantation. So far, the effect of G-CSF on natural killer (NK) cells has not been fully investigated. STUDY DESIGN AND METHODS: The effect of G-CSF on the phenotype, cytokine secretion profile, and cytotoxicity of NK cells was assessed. NK cells incubated in vitro in presence of G-CSF for 48 hours as well as NK cells isolated from peripheral blood of G-CSF-mobilized stem cell donors (in vivo) were used. RESULTS: In vitro, G-CSF caused a strongly altered phenotype in NK cells with 49% down regulation of NKp44 frequency. Furthermore, the expression of the activating receptors NKp46 and NKG2D decreased 40 and 64%, respectively. The expression of KIR2DL1 and KIR2DL2 decreased by 46% each. In cytotoxicity assays, the lytic capacity of G-CSF-exposed NK cells is reduced by up to 68% in vitro and up to 83% in vivo. Accordingly, granzyme B levels of in vivo G-CSF-exposed NK cells were reduced by up to 87% in comparison to nonstimulated NK cells. Cytokine production of in vitro and in vivo incubated NK cells was strongly decreased for interferon-γ, tumor necrosis factor-α, and granulocyte macrophage colony-stimulating factor as well as interleukin (IL)-6 and IL-8. Furthermore, we observed a reduction in proliferation and a positive feedback loop that increased the expression of the G-CSF receptor. CONCLUSION: G-CSF was demonstrated to be a strong inhibitor of NK cells activity and may prevent their graft-versus-leukemia effect after transplantation.

Neuroprotective effect of Fn14 deficiency is associated with induction of the granulocyte-colony stimulating factor (G-CSF) pathway in experimental stroke and enhanced by a pathogenic human antiphospholipid antibody.

Using a transgenic mouse model of ischemic stroke we checked for a possible interaction of antiphospholipid antibodies (aPL) which often cause thromboses as well as central nervous system (CNS) involvement under non-thrombotic conditions and the TWEAK/Fn14 pathway known to be adversely involved in inflammatory and ischemic brain disease. After 7 days, infarct volumes were reduced in Fn14 deficient mice and were further decreased by aPL treatment. This was associated with strongest increase of the endogenous neuroprotective G-CSF/G-CSF receptor system. This unexpected beneficial action of aPL is an example for a non-thrombogenic action and the double-edged nature of aPL.

Granulocyte colony-stimulating factor (G-CSF) treatment of childhood acute myeloid leukemias that overexpress the differentiation-defective G-CSF receptor isoform IV is associated with a higher incidence of relapse.

PURPOSE: This prospective, multicenter Acute Myeloid Leukemia Berlin-Frankfurt-Muenster (AML-BFM) 98 study randomly tested the ability of granulocyte colony-stimulating factor (G-CSF) to reduce infectious complications and to improve outcomes in children and adolescents with acute myeloid leukemia (AML). However, a trend toward an increased incidence of relapses in the standard-risk (SR) group after G-CSF treatment was observed. PATIENTS AND METHODS: Of 154 SR patients in the AML-BFM 98 cohort, 50 patients were tested for G-CSF receptor (G-CSFR) RNA isoform I and IV expression, G-CSFR cell surface expression, and acquired mutations in the G-CSFR gene. RESULTS: In patients randomly assigned to receive G-CSF after induction, 16 patients overexpressing the G-CSFR isoform IV showed an increased 5-year cumulative incidence of relapse (50% +/- 13%) compared with 14 patients with low-level isoform IV expression (14% +/- 10%; log-rank P = .04). The level of G-CSFR isoform IV had no significant effect in patients not receiving G-CSF (P = .19). Multivariate analyses of the G-CSF-treated subgroup, including the parameters G-CSFR isoform IV overexpression, sex, and favorable cytogenetics as covariables, revealed the prognostic relevance of G-CSFR isoform IV overexpression for 5-year event-free survival (P = .031) and the 5-year cumulative incidence of relapse (P = .049). CONCLUSION: Our results demonstrate that children and adolescents with AMLs that overexpress the differentiation-defective G-CSFR isoform IV respond to G-CSF administration after induction, but with a significantly higher incidence of relapse.

Effects of granulocyte colony-stimulating factor on the proliferation and cell-fate specification of neural stem cells.

Granulocyte-colony stimulating factor (G-CSF) is a growth factor that regulates proliferation, differentiation and survival of hematopoietic progenitor cells. There is growing evidence to suggest that G-CSF exerts a powerful neuroprotective effect in different neurological disorders. However, it has remained to be elucidated if G-CSF has a direct effect on neural stem cells (NSCs). Here, we show that G-CSF could stimulate the proliferation of NSCs and promote their differentiation in vitro. Additionally, we have shown that G-CSF-induced proliferation of NSCs is associated with phosphorylation of STAT3, and the differentiation is linked to altered expression of differentiation-related genes. Remarkably, G-CSF could not initiate the differentiation of NSCs. The added roles of G-CSF in regulating proliferation and differentiation of NSCs as shown in this study would serve as a useful reference in designing new stem cell therapy strategies for promoting brain recovery and repair.

G-CSF administration in a small-for-size liver model.

BACKGROUND: Extended liver resection is applied in the treatment of liver tumors and during living-related liver transplantation. This procedure can lead to transient, in some patients even lethal liver failure. Administration of G-CSF is associated with an increased survival after toxic liver damage. It is the aim of this study to test the effect of G-CSF administration in the rat 90% extended liver resection model. MATERIALS: Rats with and without G-CSF treatment were subjected to 90% partial hepatectomy using a mass ligation technique. Animals were sacrificed 6 hr, 24 hr, and 7 days after resection. Read-out parameters were liver damage in terms of liver enzymes and histomorphological alterations. Hepatic regeneration was determined by measuring liver weight recovery and calculating the BrdU proliferation index of hepatocytes. G-CSF-receptor expression was visualized in both groups by immunohistochemistry. Expression of lipopolysaccharide-binding-protein (LBP) mRNA was evaluated by quantitative PCR. RESULTS: Survival rate was increased in the G-CSF-treatment group. Full liver weight recovery within one week was only achieved in the treatment group and was accompanied by reduced liver damage. G-CSF-receptor upregulation subsequent to administration of G-CSF may indicate a direct receptor mediated effect of G-CSF on the liver. Upregulation of LBP mRNA in the liver of G-CSF treated animals--reported to be associated with an increased host defense--could demonstrate a mode of action of G-CSF.

[Influence of rhG-CSF on activity of sphingosine kinase in monocytes].

The aim of this research was to understand the influence of rhG-CSF on the sphingosine kinase (SphK) activity of monocytes. The peripheral blood monocytes were collected from 6 peripheral blood progenitor cell donors on the fifth day of mobilization with rhG-CSF and from 5 blood donors' buffy coats. The mRNA expressions of monocyte G-CSF receptor and SphK were tested with RT-PCR. The changes of SphK activity of monocytes were assayed after being treated with rhG-CSF. The results showed that the two kinds monocytes collected from both blood donors and peripheral blood progenitor cell donors mobilized with rhG-CSF expressed mRNA of G-CSF receptor and SphK. The SphK activity of monocytes collected from blood donors was not changed significantly after being treated with rhG-CSF (P > 0.05). The SphK activity of monocytes collected from peripheral blood progenitor cell donors transiently increased by (39.6 - 87.2)% after being treated by means of rhG-CSF (P < 0.05) without obviously dose-dependent effect. It is concluded that the SphK activity of monocytes collected from peripheral blood progenitor cell donors can be activated by rhG-CSF.

The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.

Identification of CCR2, flotillin, and gp49B genes as new G-CSF targets during neutrophilic differentiation.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates myeloid progenitor cells to proliferate and differentiate into neutrophilic granulocytes. To identify genes induced by G-CSF during neutrophil differentiation, interleukin-3-dependent murine myeloid precursor FDC-P1 cells expressing the G-CSF receptor were stimulated with G-CSF, and the gene expression profile was characterized by DNA microarray analysis. In addition to known signal transducer and activator of transcription-3 target genes, such as suppressor of cytokine signaling-3 (SOCS3), JunB, and p19(INK4D), we newly identified several G-CSF targets, including genes for the CC chemokine receptor-2 (CCR2), raft proteins flotillin-1 and flotillin-2, and immunoglobulin-like receptor gp49B. Real-time, quantitative polymerase chain reaction analyses revealed that the expression of these genes was induced in various myeloid cell lines by G-CSF. Furthermore, when HoxA9-immortalized bone marrow progenitors were induced by G-CSF to differentiate into mature neutrophils, all of these genes were strongly activated. These genes could be categorized into three groups based on their time-course of expression: immediate-early (approximately 20 min, SOCS3), mid-early (2-4 h, flotillin-1/2 and gp49B), and late (>12 h, CCR2). This suggests that different transcriptional mechanisms are involved in the regulation of these genes. We show that bone marrow neutrophils express functional CCR2, which suggest that CC chemokines may play previously unknown roles in neutrophil activation and chemotaxis.

Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor.

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappaB site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.

G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes.

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

Granulocyte colony-stimulating factor receptor signals for beta1-integrin expression and adhesion in bladder cancer.

OBJECTIVES: To examine the association of granulocyte colony-stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) expression with increased beta1-integrin expression and determine the ability of autocrine G-CSFR signaling to promote bladder cancer cell adhesion by way of beta1-integrin. beta1-integrin is expressed at higher levels in more invasive bladder carcinoma cells and participates in the process of tissue invasion. Cancer cell invasion and metastasis in some ways mimic normal neutrophil behavior. In neutrophils, G-CSF acts through its receptor to enhance adhesion and migration. A significant fraction of bladder carcinoma has been reported to express both G-CSF and G-CSFR. METHODS: We examined bladder carcinoma tissue samples obtained from segmental or radical cystectomy specimens for expression of G-CSF, G-CSFR, and beta1-integrin using reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical methods. We determined the G-CSFR-mediated beta1-integrin adhesion using a static adhesion assay and the bladder cancer cell line 5637. RESULTS: Eleven of 14 bladder cancer samples expressed G-CSFR. All 11 G-CSFR positive tumors also expressed G-CSF, and the G-CSF/G-CSFR positive tumors had elevated beta1-integrin protein levels. All but one G-CSFR negative tumor demonstrated low beta1-integrin protein levels. In four G-CSF/G-CSFR positive tumors for which distant urothelium was available for examination, G-CSF, G-CSFR, and beta1-integrin expression was also increased. In the 5637 cell line, we demonstrated G-CSFR-mediated upregulation of beta1-integrin-dependent adhesion to fibronectin and laminin. CONCLUSIONS: G-CSF/G-CSFR expression in some bladder cancers appears to be an early event during malignant transformation that increases beta1-integrin expression and adhesion and thereby may promote tissue invasion.