Highly Expressed Granulocyte Colony-Stimulating Factor (G-CSF) and Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) in Human Gastric Cancer Leads to Poor Survival.

BACKGROUND Chemotherapy for advanced gastric cancer (GC) patients has been the mainstay of therapy for many years. Although adding anti-angiogenic drugs to chemotherapy improves patient survival slightly, identifying anti-angiogenic therapy-sensitive patients remains challenging for oncologists. Granulocyte colony-stimulating factor (G-CSF) promotes tumor growth and angiogenesis, which can be minimized with the anti-G-CSF antibody. Thus, G-CSF might be a potential tumor marker. However, the effects of G-CSF and G-CSFR expression on GC patient survival remain unclear. MATERIAL AND METHODS Seventy GC tissue samples were collected for G-CSF and G-CSFR detection by immunohistochemistry. A total of 40 paired GC tissues and matched adjacent mucosa were used to measure the G-CSF and G-CSFR levels by ELISA. Correlations between G-CSF/G-CSFR and clinical characteristics, VEGF-A levels and overall survival were analyzed. Biological function and underlying mechanistic investigations were carried out using SGC7901 cell lines, and the effects of G-CSF on tumor proliferation, migration, and tube formation were examined. RESULTS The levels of G-CSFR were upregulated in GC tissues compared to normal mucosa tissues. Higher G-CSF expression was associated with later tumor stages and higher tumor VEGF-A and serum CA724 levels, whereas higher G-CSFR expression was associated with lymph node metastasis. Patients with higher G-CSF expression had shorter overall survival times. In vitro, G-CSF stimulated SGC7901 proliferation and migration through the JAK2/STAT3 pathway and accelerated HUVEC tube formation. CONCLUSIONS These data suggest that increased G-CSF and G-CSFR in tumors leads to unfavorable outcomes for GC patients by stimulating tumor proliferation, migration, and angiogenesis, indicating that these factors are potential tumor targets for cancer treatment.

Granulocyte-colony stimulating factor: a new player for the enteric nervous system.

The enteric nervous system (ENS) controls and modulates gut motility and responds to food intake and to internal and external stimuli such as toxins or inflammation. Its plasticity is maintained throughout life by neural progenitor cells within the enteric stem cell niche. Granulocyte-colony stimulating factor (G-CSF) is known to act not only on cells of the immune system but also on neurons and neural progenitors in the central nervous system (CNS). Here, we demonstrate, for the first time, that G-CSF receptor is present on enteric neurons and progenitors and that G-CSF plays a role in the expansion and differentiation of enteric neural progenitor cells. Cultured mouse ENS-neurospheres show increased expansion with increased G-CSF concentrations, in contrast to CNS-derived spheres. In cultures from differentiated ENS- and CNS-neurospheres, neurite outgrowth density is enhanced depending on the amount of G-CSF in the culture. G-CSF might be an important factor in the regeneration and differentiation of the ENS and might be a useful tool for the investigation and treatment of ENS disorders.

High-Throughput Process Development: I-Process Chromatography.

Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in the removal of product-related, host-cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during the development of a chromatographic step. Traditional process development is performed as a trial-and-error-based evaluation and often leads to a suboptimal process. A high-throughput process development (HTPD) platform involves the integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatographic process development. Creation of such platforms requires the integration of mechanistic knowledge of the process with various statistical tools for data analysis. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing the HTPD of chromatography steps. It describes the operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6 µL well size. We also discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.93). We think that this protocol will be of significant value to those involved in performing the high-throughput process development of the chromatography process.

Novel role of WD40 and SOCS box protein-2 in steady-state distribution of granulocyte colony-stimulating factor receptor and G-CSF-controlled proliferation and differentiation signaling.

Signals induced by granulocyte colony-stimulating factor (G-CSF), the major cytokine involved in neutrophil development, are tightly controlled by ligand-induced receptor internalization. Truncated G-CSF receptors (G-CSF-Rs) that fail to internalize show sustained proliferation and defective differentiation signaling. Steady-state forward routing also determines cell surface levels of cytokine receptors, but mechanisms controlling this are poorly understood. Here, we show that WD40 and suppressor of cytokine signaling (SOCS) box protein-2 (Wsb-2), an SOCS box-containing WD40 protein with currently unknown function, binds to the COOH-terminal region of G-CSF-R. Removal of this region did not affect internalization, yet resulted in increased membrane expression of G-CSF-R and enhanced proliferation signaling at the expense of differentiation induction. Conversely, Wsb-2 binding to the G-CSF-R reduced its cell surface expression and inhibited proliferation signaling. These effects depended on the SOCS box involved in ubiquitylation and on cytosolic lysines of G-CSF-R and imply a major role for ubiquitylation through the G-CSF-R C-terminus in forward routing of the receptor. Importantly, the Wsb-2 gene is commonly disrupted by virus integrations in mouse leukemia. We conclude that control of forward routing of G-CSF-R is essential for a balanced response of myeloid progenitors to G-CSF and suggest that disturbance of this balance may contribute to myeloid leukemia.

Pretreatment with granulocyte colony-stimulating factor attenuated renal ischaemia and reperfusion injury via activation of PI3/Akt signal pathway.

AIM: Granulocyte colony-stimulating factor (G-CSF) has been shown to exert protective effects in various tissues and experimental models of ischaemia-induced injury. However, the mechanism of renoprotective action in ischaemia/reperfusion (I/R) renal injury of G-CSF was unknown. METHODS: Male C57BL/6J mice, subjected to renal ischaemia for 45 min, 48 h and 7 days reperfusion, were administered either saline, wortmannin, G-CSF, and G-CSF plus wortmannin 3 days prior to I/R. Saline-treated group served as the control. At 48 h and 7 days of reperfusion, the mice were killed. RESULTS: Significantly, renal dysfunction and morphological injury were identified at 48 h and 7 days after I/R. Wortmannin pretreatment worsened the renal injury significantly. However, G-CSF pretreatment significantly attenuated renal injury, reduced the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive ratio of renal tubular epithelial cells and inflammation cytokine expression in the kidney. Moreover, G-CSF pretreatment inhibited the expression of Bax and increased the expression of bcl-2 and p-Akt in the kidney. Wortmannin blunted the beneficial effects of G-CSF. CONCLUSION: The cytoprotective action of G-CSF against I/R injury seems to be associated with its anti-apoptotic action mediated by upregulation of p-Akt signal pathway.

Therapeutic use of granulocyte-colony stimulating factor could conceal residual malignant cells in patients with AML1/ETO+ acute myelogenous leukemia.

We have experienced a number of cases of AML1/ETO+ acute myelogenous leukemia that showed remission based on bone marrow (BM) morphological criteria, but that revealed clonal abnormalities in most cells by fluorescence in situ hybridization (FISH). Interestingly, most of these cases had AML with AML1/ETO rearrangement. The malignant cells were differentiated and considered mature cells after granulocyte-colony stimulating factor (G-CSF) treatment. To clarify the possible mechanisms underlying this phenomenon, we investigated the expression levels of G-CSFR in AML cells with AML1/ETO rearrangement by flow cytometry and real-time polymerase chain reaction (PCR). The number of AML1/ETO+ cells expressing G-CSFR at baseline was significantly higher than that of AML1/ETO- AML cells (2673 vs 522). In addition, the G-CSFR gene was more highly expressed in AML1/ETO+ cells than in AML1/ETO- cells by real-time PCR. This study reveals that cases showing remission after treatment with G-CSF mostly had leukemia with AML1/ETO rearrangement. This finding might be explained by the higher expression of G-CSF receptor in AML1/ETO+ cells than in AML1/ETO- cells. We recommend that remission should be confirmed by FISH, because malignant clones can be differentiated and masked in morphological examination or chromosome test, especially for AML with AML1/ETO rearrangement.

Mobilization of bone marrow stem cells by granulocyte colony-stimulating factor ameliorates radiation-induced damage to salivary glands.

PURPOSE: One of the major reasons for failure of radiotherapeutic cancer treatment is the limitation in dose that can be applied to the tumor because of coirradiation of the normal healthy tissue. Late radiation-induced damage reduces the quality of life of the patient and may even be life threatening. Replacement of the radiation-sterilized stem cells with unirradiated autologous stem cells may restore the tissue function. Here, we assessed the potential of granulocyte colony-stimulating factor (G-CSF)-mobilized bone marrow-derived cells (BMC) to regenerate and functionally restore irradiated salivary glands used as a model for normal tissue damage. EXPERIMENTAL DESIGN: Male-eGFP+ bone marrow chimeric female C57BL/6 mice were treated with G-CSF, 10 to 60 days after local salivary gland irradiation. Four months after irradiation, salivary gland morphology and flow rate were assessed. RESULTS: G-CSF treatment induced homing of large number of labeled BMCs to the submandibular glands after irradiation. These animals showed significant increased gland weight, number of acinar cells, and salivary flow rates. Donor cells expressed surface markers specific for hematopoietic or endothelial/mesenchymal cells. However, salivary gland acinar cells neither express the G-CSF receptor nor contained the GFP/Y chromosome donor cell label. CONCLUSIONS: The results show that BMCs home to damaged salivary glands after mobilization and induce repair processes, which improve function and morphology. This process does not involve transdifferentiation of BMCs to salivary gland cells. Mobilization of BMCs could become a promising modality to ameliorate radiation-induced complications after radiotherapy.

Establishment of a leukemia cell line with i(12p) from a patient with a mediastinal germ cell tumor and acute lymphoblastic leukemia.

We report the establishment of a leukemia cell line (UoC-B10) from a patient who developed leukemia several months after the diagnosis of a mediastinal yolk sac tumor. The patient's yolk sac tumor responded to combination chemotherapy, and a mature teratoma with focal areas of hematopoiesis was subsequently resected. However, 5 months after the initial diagnosis, the patient developed an acute lymphoblastic leukemia with a precursor B-cell phenotype. Cytogenetic analysis showed an i(12p) abnormality in the patient's leukemia cells and in the UoC-B10 cell line. The i(12p) was also identified retrospectively in the mediastinal tumor cells by fluorescent in situ hybridization analysis. The UoC-B10 cell line, which has been growing continuously for > 24 months in culture, was Epstein-Barr virus negative and was generally concordant with the patient's leukemia cells by analysis of immunophenotype, karyotype, and genotype. The UoC-B10 cell line possesses receptors for granulocyte-colony-stimulating factor, a cytokine which the patient received as part of his treatment protocol. This cell line may be useful in studying the relationship between i(12p) and hematological differentiation of human mediastinal germ cell tumors.

Autocrine growth of transitional cell carcinoma of the bladder induced by granulocyte-colony stimulating factor.

Granulocyte-colony stimulating factor (G-CSF) produced by nonhematopoietic malignant cells has been reported to be capable of inducing a leukemoid reaction in the host through intense stimulation of leukocyte production. Furthermore, this is frequently associated with aggressive tumor cell growth and a detrimental clinical outcome. In this study, we identified bladder cancer cells producing G-CSF with the expression of the functional receptor, which provides direct evidence of autocrine growth of bladder cancer cells induced by G-CSF. The cancer cells used in this study were obtained from a 76-year-old man who had a metastatic transitional cell carcinoma of the bladder and who demonstrated marked leukocytosis, his peripheral blood leukocyte count was 94,900 leukocytes/mm3, and his serum G-CSF level was 103 pg/ml. The culture medium in which the cancer cells were grown exclusively contained a significant amount of G-CSF (5560 pg/ml). Significant G-CSF mRNA expression and G-CSF receptor mRNA expression in the cultured cells were demonstrated by the reverse transcription-PCR method. In addition, binding studies with the use of radiolabeled recombinant G-CSF demonstrated the presence of high-affinity G-CSF binding receptors on the cultured cancer cells. Finally, the proliferation of the cultured cancer cells was stimulated by exogenous G-CSF administration, and this stimulation was inhibited by adding anti-G-CSF antibody, as demonstrated by both the flow cytometric bromodeoxyuridine incorporation technique and the [3H]thymidine incorporation assay. These results strongly suggest that G-CSF production by the bladder cancer cells studied augments autocrine growth. Therefore, we recommend exercising caution in the clinical use of G-CSF for bladder cancer patients.

Granulocyte colony-stimulating factor receptors on human acute leukemia: biphenotypic leukemic cells possess granulocyte colony-stimulating factor receptors.

Granulocyte colony-stimulating factor (G-CSF) receptors on the gated leukemic blast cells from newly diagnosed patients with acute leukemia or crisis of chronic myelogenous leukemia were investigated using flow cytometric detection. Surface marker analysis and cytochemical studies were conducted simultaneously to characterize the blast cells. Among 24 leukemia cases examined, G-CSF receptor-positive blast cells were detected in all 11 cases of acute myeloblastic leukemia even though the percentage range of positive cells was widely variable. On the other hand, they were not detected on the blast cells from patients with peroxidase-negative acute lymphoblastic leukemia with no myeloid surface antigens. However, G-CSF receptors were demonstrated in significant amounts on blast cells from 5 of 8 cases of peroxidase-negative acute leukemia expressing both myeloid and lymphoid surface antigens (biphenotypic leukemia). The percentage of blast cells positive for G-CSF receptors was significantly smaller in biphenotypic cases [33 +/- 14% (SD)] than in acute myeloblastic leukemia cases [65 +/- 22%] (P less than 0.01). The percentage expression of CD13 antigen by blast cells was significantly related to their percentage positivity for G-CSF receptors (rs = 0.50, P less than 0.05). These findings indicate that the distribution of flow cytometrically detectable G-CSF receptors on leukemic cells possessing myeloid characteristics may be related to the maturation process.

Dysregulation of transcriptions in primary granule constituents during myeloid proliferation and differentiation in patients with severe congenital neutropenia.

We examined the expression of granule constituent genes in myeloid progenitor cells during proliferation and differentiation in patients with severe congenital neutropenia (SCN). The heterozygous mutation of the neutrophil elastase gene was identified in two of four patients. The CD34+/granulocyte-colony stimulating factor receptor (G-CSFR)+ cells of SCN patients showed defective responsiveness to G-CSF in serum-deprived culture. The CD34+/G-CSFR+ cells expressed low levels of the granule constituent mRNAs. The transcription levels of primary granule enzyme genes in CD34+/G-CSFR+ cells were gradually enhanced and then decreased when cells were induced toward myeloid lineage with G-CSF in normal subjects. However, the primary up-regulation and the following down-regulation of these enzyme transcriptions were not clearly observed in SCN patients. No differences in expressions of the lactoferrin gene were seen between normal subjects and patients with SCN. We hypothesize that the abnormal regulation of the transcription in primary granule constituents might involve the defective proliferation and differentiation of myeloid cells in patients with SCN.

Modulation of human G-CSF receptor mRNA and protein in normal and leukemic myeloid cells by G-CSF and retinoic acid.

Granulocyte colony-stimulating factor (G-CSF) is produced by several cell types throughout the body and has a variety of effects on neutrophils and their precursors, which are mediated by binding to its receptor. It is not yet known what physiologic factors modulate G-CSF receptor mRNA expression in these cells. We studied the effect of G-CSF on freshly isolated neutrophils and bone marrow cells from normal human subjects and on myeloid leukemic cell lines. We found that G-CSF receptor mRNA levels were maintained by G-CSF in neutrophils but not in bone marrow cells. Of the leukemic cell lines tested, K562 and BV173, both of which contain the bcr-abl translocation, neither expressed G-CSF receptor mRNA. Whereas G-CSF did not affect mRNA levels for its receptor in myeloid leukemic cell lines, exposure of the acute promyelocytic cell line, NB4, to all-trans retinoic acid induced a striking increase in G-CSF receptor mRNA expression and resulted in increased G-CSF receptor surface expression. The effect of retinoic acid on G-CSF receptor mRNA on NB4 cells occurred early, before morphologic evidence of differentiation, and required protein synthesis. All-trans retinoic acid also upregulated G-CSF receptor mRNA in the myeloid leukemia cell line HL-60. Thus, maintenance of G-CSF receptor on neutrophils by G-CSF may extend the duration of ligand responsiveness. Furthermore, the ability of retinoic acid to up-regulate G-CSF receptor may account for the synergistic effect of G-CSF and retinoic acid in differentiation induction of acute promyelocytic leukemia.

High-frequency granuloid colony-forming ability of G-CSF receptor possessing CD34 antigen positive human umbilical cord blood hematopoietic progenitors.

The presence of granulocyte colony-stimulating factor receptor (G-CSFR) in CD34+ hematopoietic progenitors is demonstrated by flow cytometry. Cord blood mononuclear cells (MNCs) contain about 1.09 +/- 0.2% CD34+ cells, 0.32 +/- 0.1% CD34+/G-CSFR+ (G-CSFR+) cells, and 0.77 +/- 0.1% CD34+/G-CSFR- (G-CSFR-) cells. The colony-forming ability of G-CSFR+ cells in the presence of G-CSF, granulocyte-macrophage CSF (GM-CSF), interleukin-3 (IL-3), and erythropoietin (Epo) was higher than that of G-CSFR- cells (29.5 vs. 9.8%; p < 0.01). In the fraction of G-CSFR+ cells, the most frequently formed colony type was CFU-G/GM, while burst-forming unit-erythroid (BFU-E) or colony-forming unit-macrophage (CFU-M) were rare. On the other hand, the incidence of BFU-E and CFU-G/GM was similar in the fraction of G-CSFR- cells. This indicates that most granuloid colonies of CD34+ cells were derived from G-CSFR+ cells. These results suggest a lineage commitment for the vast majority of G-CSFR+ hematopoietic progenitors.

Clonal evolution in marrows of patients with Shwachman-Diamond syndrome: a prospective 5-year follow-up study.

OBJECTIVES: Shwachman-Diamond syndrome (SDS) is characterized by varying degrees of marrow failure. Retrospective studies suggested a high propensity for malignant myeloid transformation into myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The study's aims were to determine the cellular and molecular characteristics as well as the clinical course of malignant myeloid transformation and clonal marrow disease in patients with SDS. METHODS: This is a longitudinal prospective study of 14 patients recruited for annual hematological evaluations. Results of baseline and serial hematological assessments for up to 5 years are reported. RESULTS: Clonal marrow cytogenetic abnormalities (CMCA) were detected in 4 patients (29%) on first testing or at follow-up. The abnormalities were del(20q) in two patients, i(7q) in one, and combined del(20q) and i(7q) in one. The following tests did not distinguish patients with CMCA from other SDS patients: severity of peripheral cytopenia, fetal hemoglobin levels, percentage of marrow CD34+ cells, colony growth from marrow CD34+ cells, cluster-to-colony ratio, marrow stromal function, percentage of marrow apoptosis cells, and granulocyte colony-stimulating factor receptor expression. RAS and p53 mutation analysis and AML blast colony assays were uniformly negative. No patients showed progression into more advanced stages of MDS or into AML. In one patient, the abnormal clone became undetectable after 2 years of follow-up. CONCLUSIONS: We conclude that although CMCA in SDS is high, progression into advanced stages of MDS or to overt AML may be slow and difficult to predict. Treatment should be cautious since some abnormal clones can regress.

The effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro maturation of porcine oocyte on subsequent embryonic development.

Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus-oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus-oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF-treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF-treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF-treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF-treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF-treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.

G-CSF increases secretion of urokinase-type plasminogen activator by human lung cancer cells.

We reported previously that granulocyte colony-stimulating factor (G-CSF) can promote the invasion of human lung cancer cell lines in vitro. However, the exact mechanism of its stimulatory effect on invasion remains to be elucidated. In the present study we mainly focused our attention on the components of the plasminogen activation system in human lung cancer cell lines, because of the central role that plasminogen activators play in regulating extracellular proteolysis. We showed that G-CSF induced a dose-dependent increase in the urokinase-type plasminogen activator (uPA) activity in the conditioned medium of a PC-9 lung cancer cell line. When the amounts of uPA activity were quantitated by densitometry, we found that even at a concentration of 0.01 microg/ml, G-CSF had a stimulatory effect on the uPA release, while high concentrations caused a 3.6-fold increase at a maximum concentration of 1 microg/ml. A Western blot analysis of the conditioned medium confirmed the findings observed in a zymographic analysis. The observed increase in uPA protein was paralleled by a significant increase in the uPA mRNA levels after treatment with G-CSF. However, our experiments failed to identify any alteration in the plasminogen activator inhibitor (PAI) secretion caused by G-CSF. In addition, we also found the expression of G-CSF receptor by PC-9 cells, suggesting the possible pathway activated by G-CSF.

Coexpression of granulocyte colony stimulating factor and its receptor in primary ovarian carcinomas.

Immunohistochemistry was used to determine the expression of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in primary ovarian carcinomas. The expression of G-CSFR was observed in the malignant cells of each of the 46 primary carcinomas examined; G-CSF was coexpressed in both the malignant epithelial cells and the stroma of 56.5% of the specimens. Thus the majority of ovarian carcinomas harbor both potential autocrine and paracrine G-CSF axes. In 37% of the samples, G-CSF was expressed only within stromal cells, suggesting that only a potential paracrine system is in place. In a preliminary, retrospective, evaluation, the survival of patients whose tumors expressed only the apparent paracrine loop was significantly worse than patients whose tumors expressed both potential autocrine and paracrine G-CSF-based regulatory loops (14.5 vs. 42.5 months, respectively). Studies on the potential function of G-CSF were performed using the G-CSFR-expressing OVCAR-3 ovarian carcinoma line. As a single agent, rhG-CSF failed to stimulate [3H]-thymidine incorporation in these cells, but enhanced the mitogenic action of epidermal growth factor (EGF) in a dose-dependent manner. Thus, potential autocrine and/or paracrine loops involving G-CSF and its receptor occur in over 90% of primary ovarian carcinomas, and may act to modulate the action of growth factors.

Acute myeloblastic leukemia (ANLL-M2) with t(8;21)(q22;q22) variant expressing lymphoid but not myeloid surface antigens with a high number of G-CSF receptors.

Leukemic cells from an 8-year-old girl with ANLL-M2 expressed precursor B-cell antigen CD19, but none of the myeloid antigens CD11b, CD13, CD14 and CD33. After culture, the cells expressed CD11b and CD13. The cells carried a high number of granulocyte colony-stimulating factor (G-CSF) receptors. In chromosome analysis, metaphase cells were obtained only in the case of culture with G-CSF. The karyotype was a variant of t(8;21)(q22;q22). Southern blot analysis revealed rearrangement of the AMLI gene located on chromosome 21. These observations may suggest that even without myeloid surface antigens and with precursor B-cell antigen, ANLL-M2 with t(8;21)(q22;q22) has apparent myeloid characteristics.

Analysis of myeloid characteristics in acute lymphoblastic leukemia.

We examined myeloid characteristics of myeloid-antigen-positive (My+) and -negative (My-) B-precursor acute lymphoblastic leukemia (ALL) blast cells. Immunophenotyping before and after culture, rearrangements of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes, stimulation of DNA synthesis with granulocyte colony-stimulating factor (G-CSF), and G-CSF binding assay were performed. Of My+ ALL blasts, the immunophenotypic staging as B-precursor ALL and rearrangements of IgH and TCR-beta, gamma and delta genes did not differ from findings in My- ALL blasts. Stimulated with G-CSF, cells from one My+ ALL and from one My- ALL patients showed enhancement of DNA synthesis and expression of CD11b and CD13, respectively. G-CSF binding was observed in blasts from 3 My+ ALL patients and one My- ALL child. After culture, blasts from My- ALL children expressed CD13 but showed neither enhanced DNA synthesis with G-CSF nor G-CSF binding. Thus, it would appear that (i) My+ and My- adult ALL blasts are at the same stage of differentiation; (ii) some My+ adult ALL blasts have phenotypic and functional myeloid characteristics; and (iii) induction of CD13 expression in My- ALL after in vitro culture does not correlate with other myeloid characteristics.

Effects of recombinant human granulocyte colony-stimulating factor on the growth potential of two murine myeloid leukemias.

We investigated the in vitro and in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the proliferation of two murine leukemic cell lines. The rhG-CSF stimulated leukemic colony formation of the promyelocytic leukemic cell line L-8801 in methylcellulose culture and increased the number of L-8801 cells in liquid culture. However, rhG-CSF treatment prolonged the median survival period of mice implanted with L-8801 cells and the emergence of the leukemic blast cells in peripheral blood. Meanwhile, rhG-CSF had no influence on that of the megakaryoblastic leukemic cells L-8057 and failed to prolong the median survival period of L-8057 leukemic mice. Receptor binding analysis revealed that L-8801 cells expressed a G-CSF receptor (Kd=125 pM, 479 binding sites/cell) and L-8057 cells had no G-CSF receptors. Then, we examined the growth potential of these cells. The median survival period was longer for mice implanted with L-8801 cells cultured with rhG-CSF for 72 h in vitro than for cells grown without rhG-CSF. Furthermore, the median survival period of mice implanted with spleen cells from L-8801 leukemic mice treated with rhG-CSF was prolonged compared with those from leukemic mice without rhG-CSF. In contrast, there was no effect of rhG-CSF on the growth potential of the spleen from L-8057 leukemic mice. The results of our present study demonstrate that rhG-CSF reduced the growth of L-8801 leukemic cells in vitro and in vivo mediated through G-CSF receptors, thereby suppressing the development of leukemia.