Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on lung cancer: roles of cyclooxygenase-2

We examined the effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the lung cancer cell lines PC-9, LA-1 and A549. In addition, we examined if the effects of the cytokines on the cell lines are mediated by activation of cyclooxygenase (COX)-2. The three cell lines did not constitutively produce either G-CSF or GM-CSF. G-CSF did not influence cell growth in the three cell lines, while GM-CSF increased cell growth in the A549 and LA-1 lines. G-CSF and GM-CSF dose-dependently decreased cell death in the three cell lines. RT-PCR demonstrated GM-CSF receptor expression in the three lung cancer cell lines, whereas the G-CSF receptor exists only in the PC-9 line. We suggest that G-CSF might rescue the tumor cells from cytotoxicity due to serum deprivation through cellular pathways independent of the G-CSF receptor. G-CSF and GM-CSF increased cyclooxygenase-2 (COX-2) expression in PC-9 and LA-1 cells whereas they decreased COX-2 expression in A549 cells. The COX-2 inhibitor NS-398 increased cell death in PC-9 and LA-1 cells, whereas it decreased cell death in A549 cells. PC-9 and LA-1 clones transfected with sense G-CSF- or GM-CSF showed an increase in COX-2 expression, while COX-2 expression was decreased in transfected A549 clones. COX-2 expression was increased in anti-sense G-CSF- and GM-CSF-transfected A549 clones. Thus, although COX-2 activation seems to induce different biological behavior depending on the cell type, we propose that G-CSF and GM-CSF might accelerate tumor progression by directly regulating COX-2 expression, independently of an autocrine mechanism.     

Role of autocrine and paracrine production of granulocyte-macrophage colony-stimulating factor and interleukin-1 beta in the autonomous growth of acute myeloblastic leukaemia cells--studies using purified CD34-positive cells.

The blast cells from some patients with acute myeloblastic leukemia (AML) proliferate autonomously in vitro. We have previously identified four groups of AML blasts based upon their growth characteristics in vitro, in particular the degree of autonomous growth. We have now measured the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-1 beta (IL-1 beta) by AML cells with different growth characteristics, using two sensitive enzyme-linked immunosorbent assays. Our results show a correlation between the capacity of AML blasts to produce GM-CSF and IL-1 beta and the ability to grow autonomously in vitro. Blasts from cells with no autonomous growth (n = 5) secreted low or undetectable amounts of GM-CSF and IL-1 beta. Blasts with totally autonomous growth (n = 10) secreted the highest levels of GM-CSF (mean 2469 pg/10(3) cells) and IL-1 beta (mean 3156 pg/10(6) cells). Whereas blasts with partially autonomous growth (n = 9) secreted intermediate levels of GM-CSF (mean 270 pg/10(6) cells) and IL-1 beta (mean 931 pg/10(6) cells). In order to determine whether GM-CSF production was autocrine or the consequence of paracrine secretion by differentiated leukemic cells, we studied the degree of autonomous growth and production of GM-CSF by CD34-positive blasts from eight patients whose unfractionated cells produced GM-CSF. We found that CD34-positive blasts from six of these cases grew autonomously to a degree comparable to that of the unfractionated cells, and that CD34-positive blasts produced GM-CSF either autonomously or in response to recombinant IL-1 beta. Our data suggests that in the majority of cases, CD34-positive blasts are capable of autonomous growth and autocrine GM-CSF production, however this is variably regulated by the paracrine production of IL-1 beta by CD34-negative cells.     

Effect of protein kinase inhibitors on the proliferation of leukemic cells stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor or interleukin-3.

The effects of the inhibitor for protein kinase A or C, or tyrosine kinase (H-8, staurosporine, or genistein, respectively) on the proliferation of leukemic and normal bone marrow cells stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3) were studied using the MTT assay. These inhibitors suppressed the proliferation of leukemic and normal bone marrow cells in a dose-dependent manner. Although the suppressive effect of each inhibitor on cell proliferation was varied in each instance, the effects were almost similar whichever CSF was added. A significant difference was not recognized between leukemic and normal bone marrow cells in terms of sensitivity to these inhibitors. The data indicate that protein kinase inhibitors have an inhibitory effect on leukemic and normal hematopoietic cell proliferation and that further studies are required to determine if this effect is due to the inhibition of protein kinases acting as the second messenger of CSFs.     

Hematologic effects of interleukin-1 beta, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor in tumor-bearing mice treated with fluorouracil.

Myelosuppression following intensive chemotherapy in cancer patients is associated with increased morbidity and mortality. Hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), alone or in combination with interleukin-1 (IL-1), have been shown to counteract myelosuppression resulting from some, but not all, chemotherapeutic regimens. In an attempt to apply these findings to intensive therapy with proliferation-dependent chemotherapeutic drugs such as fluorouracil (5-FU), we investigated combination biochemotherapy in a murine model. Female CD8F1 [(BALB/c X DBA/8)F1] mice bearing first-passage transplants of spontaneous CD8F1 breast tumors were treated intraperitoneally once a week for 3 successive weeks with a course of 5-FU alone or with a course of 5-FU in combination with recombinant human interleukin-1 beta (rHuIL-1 beta) alone or in combination with CSFs. rHuIL-1 beta alone or in combination with rHuG-CSF or recombinant murine GM-CSF significantly improved tumor growth inhibition (60% vs. 90%) and survival (20% vs. 90%-100%), increased the maximally tolerated dose of 5-FU, accelerated recovery of neutrophil counts in peripheral blood, and reduced duration of significant neutropenia and loss of body weight (29% vs. 10% loss). Clinical trials of IL-1 have been initiated in patients with advanced cancer receiving multiple courses of high-dose 5-FU.     

Effect of human recombinant granulocyte/macrophage colony-stimulating factor and native granulocyte colony-stimulating factor on clonogenic leukemic blast cells

The effects of human recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and human native purified granulocyte colony-stimulating factor (G-CSF) on the growth of clonogenic leukemic blast cells from eight Japanese patients with acute myeloblastic leukemia were studied, using an in vitro leukemic blast colony assay. The results showed that GM-CSF stimulated leukemic blast colony formation in all cases examined, whereas G-CSF stimulated colony formation in four of the eight cases. The maximum stimulating activity of GM-CSF on the growth of clonogenic leukemic blast cells was higher than that of G-CSF in the majority of cases, while sometimes GM-CSF and G-CSF worked synergistically. Thus, the clonogenic leukemic blast cell populations seemed to be heterogeneous with respect to their in vitro response to growth regulators.     

Opposite effect of tumor necrosis factor alpha on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors.

The effect of recombinant human tumor necrosis factor alpha (TNF-alpha) on normal and chronic myeloid leukemia granulocyte-macrophage progenitors (CFU-GM) growing in semisolid agar cultures in the presence of recombinant granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor was studied. Granulocyte-macrophage colony-stimulating factor-dependent growth of normal and chronic myeloid leukemia bone marrow CFU-GM was greatly enhanced by TNF-alpha at doses of 0.1 to 100 units/ml. Growth enhancement included neutrophil, eosinophil, and monocyte-macrophage colonies and clusters at 7 and 14 days of culture. Since similar results were achieved with highly enriched progenitor cell populations, devoid of accessory cells, an indirect effect on CFU-GM growth through the release by accessory cells of other cytokines upon TNF-alpha stimulation was thus ruled out. By contrast, the same doses of TNF-alpha inhibited the growth of normal CFU-GM in granulocyte colony-stimulating factor-dependent cultures. Taken together, our findings indicate that the final effect of TNF-alpha on normal bone marrow granulocyte-macrophage progenitor growth is dependent on the specific growth factor interacting with it, and that both normal and chronic myeloid leukemia CFU-GM are equally responsive to the combined effects of TNF-alpha and a given colony-stimulating factor.     

Clinical role of granulocyte-macrophage colony-stimulating factor

The activity of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) for increasing leukocyte production and enhancing mature neutrophil function has been clearly demonstrated in phase I clinical trials in patients with a variety of hematologic and malignant diseases. The focus of current studies includes determination of optimal administration schedules and its use in combination with myelosuppressive chemotherapy, radiation therapy or antimicrobial agents. The utility of GM-CSF as a stimulant of host defense against infections and tumors is also under study.     

Prophylactic granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor decrease febrile neutropenia after chemotherapy in children with cancer: a meta-analysis of randomized controlled trials.

PURPOSE: To determine whether prophylactic hematopoietic colony-stimulating factors (CSFs) used in children with cancer reduce the rate of febrile neutropenia, hospitalization duration, documented infection rate, parenteral antibiotic duration, amphotericin B use, or infection-related mortality. METHODS: We included studies in this meta-analysis if their populations consisted of children, if there was randomization between CSFs and placebo or no therapy, if CSFs were administered prophylactically (before neutropenia or febrile neutropenia), and if chemotherapy treatments preceding CSFs and placebo or no therapy were identical. From 971 reviewed study articles, 16 were included. RESULTS: The mean rate of febrile neutropenia in the control arms was 57% (range, 39% to 100%). Using a random effects model, CSFs were associated with a reduction in febrile neutropenia, with a rate ratio of 0.80 (95% CI, 0.67 to 0.95; P =.01), and a decrease in hospitalization length, with a weighted mean difference of -1.9 days (95% CI, -2.7 to -1.1 days; P <.00001). CSF use was also associated with reduction in documented infections (rate ratio, 0.78; 95% CI, 0.62 to 0.97; P =.02) and reduction in amphotericin B use (rate ratio, 0.50; 95% CI, 0.28 to 0.87; P =.02). There was no difference in duration of parenteral antibiotic therapy (weighted mean difference, -4.3; 95% CI, -10.6 to 2.0 days; P =.2) or infection-related mortality (rate ratio, 1.02; 95% CI, 0.34 to 3.06; P =.97). CONCLUSION: CSFs were associated with a 20% reduction in febrile neutropenia and shorter duration of hospitalization; however, CSFs did not reduce infection-related mortality.     

Granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor in the treatment of acute myeloid leukemia and acute lymphoblastic leukemia

The role of G-CSF, GM-CSF and M-CSF in the treatment of AML and ALL was reviewed. These CSFs significantly accelerate the neutrophil recovery after intensive chemotherapy, and reduce febrile neutropenia and documented infections. There is no clear evidence that CSFs accelerate early regrowth of AML cells at the doses and schedules presently used clinically except one study. Patients who have received CSFs tend to have a higher CR rate, which does not seem to be translated into definite survival benefit. There has been no prospective randomized study showing any beneficial priming effect of CSFs on AML cells with better clinical outcomes.     

Production of granulocyte colony-stimulating factor by acute myelomonocytic leukemia cells

Two patients with acute myelomonocytic leukemia (AMMoL) were tested to determine whether or not their cells produced granulocyte colony-stimulating factor (G-CSF). Using the NFS-60 bioassay method, relatively high levels of G-CSF were demonstrated in the serum pre-treated with acid-dialysis and in the media conditioned by leukemia cells. Northern blot analysis for G-CSF using cDNA as a probe revealed that G-CSF production was increased at the mRNA level in the cells. No rearrangement at the DNA level, however, was observed by genomic Southern analysis. Our data indicate that the leukemic cells in AMMoL probably have the capacity to synthesize and secrete G-CSF.     

TGF alpha stimulates growth of skin papillomas by autocrine and paracrine mechanisms but does not cause neoplastic progression

To investigate the role of transforming growth factor alpha (TGF alpha) in tumor development, we introduced the human TGF alpha (hTGF alpha) cDNA into cultured primary mouse epidermal cells or papilloma cells using a replication-defective retroviral vector and analyzed skin grafts constructed with such cells. Expression of the exogenous gene was confirmed by detection of hTGF alpha mRNA by northern RNA blot analysis, and the secreted hTGF alpha was measured by ELISA of culture supernatants. Tumor cells expressing hTGF alpha produced benign tumors (papillomas), which were 1.5- to 2-fold larger than tumors of parental cells when tested as skin grafts on nude mice. Grafts of normal cells that expressed hTGF alpha produced normal skin. When mixtures of parental tumor cells and normal mouse keratinocytes were grafted to nude mice, papilloma formation was suppressed and tumors that did form were small. Grafts of hTGF alpha-producing papilloma cells combined with either normal epidermal cells or hTGF alpha-producing epidermal cells yielded large tumors. Mixed grafts containing keratinocytes expressing hTGF alpha and parental papilloma cells also produced large tumors. While the tumor size was substantially increased by hTGF alpha expression, the tumors that developed in all groups were histologically benign and reached a stable size 4-5 wk after grafting. These results indicate that expression of hTGF alpha by either tumor cells (autocrine) or adjoining normal cells (paracrine) can stimulate tumor growth, particularly when tumor growth is suppressed by normal tissue. However, expression of this growth factor did not appear to influence tumor progression directly.     

Randomized cross-over trial of progenitor-cell mobilization: high-dose cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) versus granulocyte-macrophage colony-stimulating factor plus G-CSF.

PURPOSE: Patient response to hematopoietic progenitor-cell mobilizing regimens seems to vary considerably, making comparison between regimens difficult. To eliminate this inter-patient variability, we designed a cross-over trial and prospectively compared the number of progenitors mobilized into blood after granulocyte-macrophage colony-stimulating factor (GM-CSF) days 1 to 12 plus granulocyte colony-stimulating factor (G-CSF) days 7 to 12 (regimen G) with the number of progenitors after cyclophosphamide plus G-CSF days 3 to 14 (regimen C) in the same patient. PATIENTS AND METHODS: Twenty-nine patients were randomized to receive either regimen G or C first (G1 and C1, respectively) and underwent two leukaphereses. After a washout period, patients were then crossed over to the alternate regimen (C2 and G2, respectively) and underwent two additional leukaphereses. The hematopoietic progenitor-cell content of each collection was determined. In addition, toxicity and charges were tracked. RESULTS: Regimen C (n = 50) resulted in mobilization of more CD34(+) cells (2.7-fold/kg/apheresis), erythroid burst-forming units (1.8-fold/kg/apheresis), and colony-forming units-granulocyte-macrophage (2.2-fold/kg/apheresis) compared with regimen G given to the same patients (n = 46; paired t test, P<.01 for all comparisons). Compared with regimen G, regimen C resulted in better mobilization, whether it was given first (P =.025) or second (P =.02). The ability to achieve a target collection of > or =2x10(6) CD34(+) cells/kg using two leukaphereses was 50% after G1 and 90% after C1. Three of the seven patients in whom mobilization was poor after G1 had > or =2x10(6) CD34(+) cells/kg with two leukaphereses after C2. In contrast, when regimen G was given second (G2), seven out of 10 patients failed to achieve the target CD34(+) cell dose despite adequate collections after C1. Thirty percent of the patients (nine of 29) given regimen C were admitted to the hospital because of neutropenic fever for a median duration of 4 days (range, 2 to 10 days). The higher cost of regimen C was balanced by higher CD34(+) cell yield, resulting in equivalent charges based on cost per CD34(+) cell collected. CONCLUSION: We report the first clinical trial that used a cross-over design showing that high-dose cyclophosphamide plus G-CSF results in mobilization of more progenitors then GM-CSF plus G-CSF when tested in the same patient regardless of sequence of administration, although the regimen is associated with greater morbidity. Patients who fail to achieve adequate mobilization after regimen G can be treated with regimen C as an effective salvage regimen, whereas patients who fail regimen C are unlikely to benefit from subsequent treatment with regimen G. The cross-over design allowed detection of significant differences between regimens in a small cohort of patients and should be considered in design of future comparisons of mobilization regimens.     

Biosynthetic granulocyte-macrophage colony-stimulating factor enhances neutrophil cytotoxicity toward human leukemia cells

Purified biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances antibody-dependent cell-mediated cytotoxicity (ADCC) of human neutrophils toward human promyelocytic leukemia cells (HL-60), B-lymphoma cells, and human T-leukemia virus II-infected human B-lymphoblastoid cells. The stimulation of antibody-dependent cell-mediated cytotoxicity is rapid (less than an hour), occurs at picomolar concentrations of GM-CSF, and does not require the presence of GM-CSF during the killing reaction. Therefore, neutrophils may be targeted toward tumor cells by antibody and their tumoricidal activity enhanced by GM-CSF in vitro. These results suggest that GM-CSF may have therapeutic utility in cancer therapy by increasing the number and activity of effector cells directed toward tumors by receptors to the immunoglobulin Fc fragment.     

Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor promote malignant growth of cells from head and neck squamous cell carcinomas in vivo

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are used to ameliorate cancer therapy-induced neutropenia and mucositis. Yet, first data in head and neck squamous cell carcinoma (HNSCC) indicate an impaired long-term prognosis on G-CSF treatment, and previous studies showed a contribution of both factors to the progression of human epithelial tumors. Therefore, we investigate the role of G-CSF and GM-CSF in progression of tumor cells from human HNSCC. Both factors stimulated proliferation and migration of tumor cell lines established from patient tumors expressing G-CSF and GM-CSF and/or their receptors. Blockade of G-CSF and GM-CSF inhibited tumor cell invasion in a three-dimensional organotypic culture model. The contribution of both factors to tumor malignancy was further confirmed in nude mouse transplants in vivo. Invasive and malignant growth yielding a similar tumor phenotype as the original patient tumor was exclusively observed in G-CSF- and GM-CSF-expressing tumors and was associated with enhanced and persistent angiogenesis and enhanced inflammatory cell recruitment. Although factor-negative tumors grew somewhat faster, they were characterized by lack of invasion, reduced and transient angiogenesis, and large necrotic areas. These data provide evidence for a progression-promoting effect of G-CSF and GM-CSF in human HNSCC and suggest further detailed evaluation of their use in the therapy of these tumors.     

Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop

Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.     

A double-blind placebo-controlled study with granulocyte-macrophage colony-stimulating factor during chemotherapy for ovarian carcinoma

In a placebo-controlled double-blind dose-finding trial, 15 patients with ovarian cancer stage III or IV received daily s.c. 1.5, 3, or 6 micrograms/kg recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). At each dose step three patients received recombinant human GM-CSF, and two received placebo. Chemotherapy comprised 6 cycles of carboplatin, 300 mg/m2, and cyclophosphamide, 750 mg/m2, by i.v. bolus on day 1 every 4 weeks. GM-CSF, given on days 6-12 on an outpatient basis, raised the mean leukocyte count on days 7, 10, and 15 and the mean neutrophil count on days 7 and 10 at all dose levels as compared with the control group. Neutrophil counts of less than 0.5 x 10(9)/liter occurred in 20 of 22 cycles in the control group and in 5 of 17 cycles at the 6-micrograms/kg/day GM-CSF dose level (P less than 0.0005). In comparison with the control group, the mean eosinophil count was higher on days 10 and 15 at all GM-CSF doses, as was the mean monocyte count on day 15. The mean platelet count was raised at the 3- and 6-micrograms GM-CSF doses on days 15 and 22. Chemotherapy dose reduction or postponement due to myelotoxicity occurred in 9 of 28 cycles in the placebo groups versus 5 of 44 cycles in the GM-CSF group (not significant). Local skin infiltrates at the GM-CSF injection sites occurred in 8/9 patients, leading to premature removal of two patients from the study. Capillary leakage of 131I-albumin was increased in all patients 5 days after the first chemotherapy course but was not significantly affected by 4 days of GM-CSF treatment. Tumor necrosis factor alpha and C-reactive protein serum levels increased during GM-CSF administration at the 6-micrograms dose level, but interleukin 6 serum levels were not affected. We conclude that a dose of 3 and 6 micrograms/kg/day GM-CSF reduces the severity of neutropenia and thrombocytopenia after carboplatin-cyclophosphamide. This GM-CSF dose does not induce additional capillary leakage.     

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.     

粒细胞-巨噬细胞集落刺激因子联合放疗研究进展

粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor,GM-CSF）作为一种造血因子可以有效诱导多种具有抑瘤效应的免疫细胞增殖,从而发挥抗肿瘤免疫反应。放疗作为肿瘤治疗的主要手段之一,不仅可以直接杀伤肿瘤细胞,而且会对抗肿瘤免疫产生影响。多项临床研究提示,放疗联合GM-CSF可以诱导产生旁观者效应并增强对肿瘤的远期控制,从而增强放疗的抗肿瘤效应。本文就GM-CSF及GM-CSF联合放疗的研究进展予以综述。      

Stimulation of the metastatic properties of Lewis-lung-carcinoma cells by autologous granulocyte-macrophage colony-stimulating factor.

Using both polymerase-chain-reaction analysis and the soft-agar colony-forming unit assay, granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to be expressed by cloned metastatic Lewis-lung-carcinoma (LLC-LN7) cells but not by non-metastatic LLC-C8 cells. Furthermore, the metastatic LLC-LN7 cells were shown to respond both to autologous GM-CSF and to exogenous recombinant GM-CSF (rGM-CSF). In the presence of neutralizing anti-GM-CSF antibodies, the metastatic LLC cells became less able to migrate or to adhere and invade through a reconstituted basement membrane. Moreover, the addition of rGM-CSF further enhanced the capacity of the metastatic LLC cells to adhere to the reconstituted basement membrane. This stimulation of metastatic properties of the LLC cells by either autologous or exogenous GM-CSF was associated with enhanced endogenous protein phosphorylation. Two proteins of approximately Mr 45,000 and Mr 64,000 were the dominant target proteins to be phosphorylated by the presence of GM-CSF. These results suggest that autologous GM-CSF may function as an autocrine stimulator of the metastatic properties of metastatic LLC cells.     

Regulation of granulocyte and macrophage colony-stimulating factor production by phytohaemagglutinin-treated blood mononuclear cells

The colony-stimulating activity (CSA) of medium conditioned by phytohaemagglutinin (PHA)-stimulated blood mononuclear cells was tested using granulocyte-macrophage colony-forming cells (GM-CFC) from normal bone marrow. Low concentrations of the conditioned medium stimulated granulocytic colony-forming cells (CFC) which formed colonies by the seventh day of incubation; higher concentrations stimulated the formation of macrophage colonies which were not seen until the end of the second week in culture.The colony-stimulating activities could not be demonstrated in adherent cell-depleted bone marrow cultures. This dependence of activity on adherent cells was confirmed by incubating different concentrations of conditioned medium with isolated adherent cells and then testing for colony-stimulating activity in cultures of non-adherent bone marrow cells. The activities of conditioned media following exposure to adherent cells corresponded to the results seen when the conditioned medium from PHA-stimulated mononuclear cell cultures was used to stimulate agar cultures of unseparated marrow. The results suggest that PHA-responsive mononuclear cells (probably T lymphocytes) may modulate the regulation of colony-stimulating factor (CSF) production by adherent colony-stimulating cells (CSC).