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Uwe M. H. Schrell, M.D., Uwe Koch, Ph.D., Rolf Marschalek, Ph.D., Thomas Schrauzer, M.D., Marc Anders, M.D., Eric Adams, Ph.D., and Rudolf Fahlbusch, M.D.
Departments of Neurosurgery, Dermatology, and Genetics, University of Erlangen-Nürnberg, Erlangen, Germany
It has been demonstrated that growth of cerebral meningiomas found in humans is controlled by a variety of factors, including growth factors, aminergic agents, neuropeptides, and steroids. The authors investigated the presence and function of the cytokines leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and oncostatin M (OSM) on meningioma cell proliferation.
Active transcription of LIF, IL-6, OSM, their related receptors (LIF-R, IL-6-R, gp130), and the consecutive signal-transducing molecules (STAT 1, STAT 3, and STAT 5a) were analyzed in reverse transcriptasepolymerase chain reaction experiments.
The presence of endogenous LIF, IL-6, and OSM proteins was demonstrated in the supernatant of cultured meningioma cells using enzyme-linked immunosorbent assay and Western blot experiments, thus indicating an autocrine signaling pathway for all three cytokines.
The biological function of all three cytokines was evaluated by studying their effects on meningioma cell growth. Recombinant LIF and IL-6 showed no significant growth modulating effects; however, recombinant OSM decreased meningioma cell growth by 66%. The antiproliferative potency of OSM was demonstrated by cell count experiments, [3H]thymidine incorporation assay, and cell cycle analysis. These in vitro data support the concept that growth of meningioma cells may be modulated by cytokines and also indicates that recombinant OSM may be one of the future candidates for use in the adjuvant treatment of inoperable and recurrent meningiomas.
Key Words * meningioma * leukemia inhibitory factor * interleukin-6 * oncostatin * receptor * growth inhibition
Benign or malignant meningiomas that have been incompletely resected have always presented a serious therapeutic problem.[55] During the last decade numerous studies were conducted using growth factors,[2,6,29,39,56,61,66,68] amines,[54,58] neuropeptides,[36,37] and steroids and their antagonists[9,19,29,35,39,40,47,48,53] to develop adjuvant therapies.
The cytokines oncostatin M (OSM),[38] leukemia inhibitor factor (LIF),[17,20,34,46] and interleukin-6 (IL-6)[21,23,24,41,72] are structurally related and perform common biological functions.[14,15,22,24,27,51,59] Primarily, they may be characterized as products of T lymphocytes,[8] which are able to induce the monocytic (macrophage) differentiation pathway in the murine leukemic myeloid cell line M1. These cytokines facilitate the release of acute-phase proteins by hepatocytes and related cells and modulate the trophic activity of neuronal cells,[4,20,30,44] however, secretion of these cytokines is not limited to cells of the hematopoietic system. Different cell types, such as human endothelial cells,[42] thymic epithelial cells,[31] brain astrocytes,[33] microglia,[33] and human fibroblasts,[11] as well as neoplastic cells from bladder cancer,[45] renal carcinoma cells,[27] and two breast carcinoma cell lines,[10] T47D and MCF-7, all secrete IL-6, LIF, or both. Recently, meningiomas were shown to secrete IL-6.[7,57,63,65] The cytokine OSM has been found in activated cells of the hematopoietic system.[38,71] All three cytokines act through a distinct receptor pathway,[16,18,69] and influence normal and neoplastic cell growth. Recombinant (r)OSM leads to inhibition of cell proliferation and to morphological changes in normal endothelial cells[50] and various tumor cell lines, such as the human melanoma cell line A-375.[26]
The objective of the current study was to analyze the biological effects of OSM, LIF, and IL-6 on meningioma cells. We also analyzed the presence of endogenous LIF, IL-6, OSM, their related receptors, and the active transcription of downstream signaling transcription factors (STAT 1, STAT 3, and STAT 5). These data indicate that rOSM is an antiproliferative agent for meningioma cells in vitro.
MATERIAL AND METHODS
Specimen Selection
Tumor specimens obtained in 10 patients (seven women, three men) with cerebral meningiomas were used in this study. All tumors were located intracranially, four were recurrent meningiomas, eight were World Health Organization[74] (WHO) Grade I and two were WHO Grade II. All patients received dexamethasone before, during, and after surgery.
Cell Culture Studies
Freshly resected meningioma tissue was washed with phosphate-buffered saline (PBS) containing 200 U/ml penicillin and 200 µg/ml streptomycin. The washed tissue was cut into pieces smaller than 1 mm3 and transferred to 75-cm2 tissue culture flasks together with growth medium (RPMI 1640) containing 5% vol/vol fetal calf serum (FCS), nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were cultured in a nonhumidified incubator at 36.7šC with 6% CO2. Cells were grown for several days and media were changed periodically. When confluence was achieved, cells were removed by adding trypsin/ethylenediamine tetraacetic acid (EDTA) (4 ml, 0.2% water/vol in 0.02% EDTA). After washing and resuspending the cells in growth medium, aliquots (105 cells/flask) were seeded into 25-cm2 tissue culture flasks.
Growth Experiments
After a 48-hour incubation period (lag phase), the medium was completely removed and 4 ml fresh growth medium (5% FCS, heat-inactivated medium, or ultraculture medium) was added to the meningioma cells with varying doses (10 pg100 ng) of rOSM, rLIF and rIL-6. Parallel experiments were conducted with neutralizing monoclonal antibodies against LIF and IL-6 and polyclonal antibodies directed against IL-6 receptor (R) protein. Cells were grown for 5 to 9 days. The experiments were ended when control cells reached near confluency. On the final day of culture, the number of cells in each flask was determined by lysing the cells (using Hepes and Zaponin buffers) and counting the nuclei with a Coulter counter. Experiments were performed in triplicate.
[3H]Thymidine Uptake
After a lag phase of 48 hours, the growth medium containing 5% FCS was discarded and replaced by serum-free medium or 5% FCS. Both the cytokines and [3H]thymidine (1 µCi/ml; 4 µCi/flask) were added to the cultures. The cells were incubated for defined time periods (630 hours) with rOSM, rLIF, or rIL-6 in concentrations of 10 pg to 100 ng. In one-half of the experimental setup, the media, including recombinant cytokines, were changed every 6 hours to reduce any action of the endogenously secreted cytokine. Experiments were performed in triplicate. At the end of the experiment the cells were washed twice in PBS and lysed with Hepes and Zaponin buffers. Deoxyribonucleic acid was precipitated at 4šC with 10% w/v tricloroacetic acid (TCA). The precipitates were transferred to 0.45 µm nitrocellulose filters, washed twice with 5% TCA at 4šC and dried overnight at 37šC in an incubator. Filters were then dissolved in 10 ml scintillation liquid and counted in a ß-counter.
Flow Cytometric Analysis of DNA
For the DNA flow cytrometric analysis, cultures were treated with rOSM (in concentrations ranging between 1 ng/ml and 100 ng/ml) under the same conditions as described in Growth Experiments. At the end of the incubation period, treated and untreated cell suspensions were washed twice with PBS and enzymatically generated by adding 3 ml 0.5% Pepsin-HCl (pH 1.5) and continuously vortexed for 15 minutes at 37šC. Stoichiometric DNA fluorochroming of the nuclei was achieved by transferring the cells into a staining solution of ethidium bromide (12.5 µg/ml) and mithramycin (25 µg/ml), dissolved in Tris-buffer (pH 7.6) admixed with 250 µl of 0.1% ribonuclease.[25] The number of stained cells corresponded to the number of cells estimated in the cell counter, approximately 105 to 106 cells/flask. After 10 minutes the fluorochromed single-strand nuclear DNA was measured with a mercury arc lamp-adapted flow cytometer and the fluorescence signals emitted were sorted via analog digital conversion and stored in a multichannel cytoanalyzer according to their intensity. The cell cycle stage distribution (percentage of cells in G0/G1, S, G2/M phases) was calculated using the planimetric method. The assumptions of this method are that: 1) the cell cycle model reflects mainly four phases; 2) a possible error factor of measurement is normally distributed; 3) the coefficient of variation is linear with respect to the DNA content measured; and 4) the S-phase density can be described by a polynomial of fixed degree.[26]
Enzyme-Linked Immunosorbent Assay of OSM, LIF, and IL-6
To determine the amount of LIF and IL-6 secreted into the culture medium, meningioma cells from five patients were set up in parallel to evaluate cell number and the cytokines on Days 2, 5, 7, and 9. On Day 5, the media were changed in all of the remaining cultures. The enzyme-linked immunosorbent assays (ELISAs) were performed according to the manufacturer's instructions. This assay system used monoclonal antibodies against OSM, LIF, and IL-6. In the case of IL-6 we diluted the media samples 1:6 and in the case of LIF and OSM the culture media were concentrated 10- and 26-fold, respectively, using Centricon 10 columns with an exclusion of Mr 10,000. In addition, we measured samples of 10- and 26-fold concentrated FCS to exclude external OSM, LIF, and IL-6 molecules.
Western Blot Analysis
To evaluate whether the expressed cytokines revealed the known molecular weight fraction (kilodalton) we collected conditioned medium from spontaneously growing meningioma cultures. Interleukin-6 was concentrated 10-fold and LIF 100-fold with Centricon 10 columns. Protein concentrations were determined using the method of Bradford. Twenty micrograms of protein from each sample was loaded onto a 10% polyacrylamide gel and subsequently transfered to a nitrocellulose membrane. The filter was blocked overnight with 2% bovine serum albumin (BSA), and washed three times with PBS 0.05% Tween 20 (PBS/TW). After a 1-hour incubation with a neutralizing antihuman OSM, LIF, or IL-6 monoclonal antibody (1:500 diluted in PBS/TW), the filter was washed four times with PBS/TW. After an additional hour of incubation with alkaline phosphataseconjugated rabbit antimouse antibody (1:1000 in PBS/TW) and three washes in PBS/TW, the filter was stained with chromogen.
Specificity Control
To confirm that the cultured meningioma cells still retained the histopathological characteristics of the primary tumor tissue, cells were grown on plastic coverslips parallel to the experiments and classified at the end of each experiment in each case as meningiomatous tissue by conventional histopathological and immunocytochemical staining.
Cell Culture and Extraction of Messenger RNA
Minced fresh meningioma tissue was transferred into 75-cm2 tissue culture flasks and passaged one or two times until none of the contamining blood cells and initial minced meningioma tissue was present in the grown monolayer. When cells were grown to near confluency (approximately 5 X 107 cells), messenger (m)RNA was extracted with the QuickPrep micro mRNA purification kit according to the manufacturer's instructions. Cells were washed three times with ice-cold PBS and lysed with extraction buffer. Isolation of Poly(A)+ mRNA was gained using oligo(dT) cellulose columns. Proteins, DNA, membranes, and cell debris were washed off with low-salt buffer and then the mRNA was eluted in the absence of salt with prewarmed elution buffer (65šC) in a volume of 0.4 ml. The final yield of approximately 2 µg mRNA was estimated by spectrophotometry at 260 nm. To minimize the likelihood of contaminating the DNA, the purified mRNA was treated with 10 U RNase-free DNase I for 60 minutes at 42šC using 50 ng human placenta DNA as a control to ensure complete digestion by DNase I. The mRNA was precipitated overnight in 95% ethanol chilled to 20šC and dissolved in a final volume of 10 µl diethy-thyro-carbonatetreated RNase-free water.
Control tissue was grown on chamber slides using conventional histological procedures to verify that the monolayer was composed of meningioma cells.
Reverse TranscriptasePolymerase Chain Reaction and Sequencing Products
Complementary (c)DNA was synthesized by a primer extension/reverse transcriptase (RT) reaction using 2 µg of Poly(A)+ mRNA from 5 X 107 cultured meningioma cells and 1 µg of oligo(dT) primer. Alternatively, 20 pmol of the downstream primer was used in the RT reaction to increase the specifity. The assays were set up in 20-µl reactions containing 5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 10 mM Tris-HCl (pH 8.8), 1 mM of each deoxynucleotide (dNTPs), 20 U ribonuclease inhibitor, 2 µg of Poly(A)+ mRNA and, 15 U of avian myeloblastosis virus RT. Complementary DNA synthesis was conducted for 1 hour at 42šC. The RT was inactivated for 5 minutes at 95šC. The first-strand cDNA reaction (20 µl) was placed on ice, diluted up to 100 µl (final buffer concentration: 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, and 1% Triton X-100), aportioned into aliquots and used for further processing or frozen at 80šC.
Cytokines and Related Receptors
Polymerase chain reaction (PCR) primers for the cytokines and related receptors were taken from literature or designed by a computerized program (Oligo 4.0, Sweden). They were commercially synthesized and purified by high-performance liquid chromatography. For amplification of RNA derived from cytokines (OSM, LIF, and IL-6) and cytokine receptors (gp130, LIF-R, and IL-6-R) we used the following oligo primers:
OSM S = 5´GAC TGG CCG ACT TAG AGC 3´; 18 mer;
OSM A = 5´GCA TGA AGC GAT GGT AGC 3´; 18 mer;
The primer pair amplifies a 282-bp cDNA fragment of the OSM gene.[38]
LIF S = 5´GAT GAG TGG AAG ATA GAG AGG 3´; 21 mer;
LIF A = 5´CCT GAC CCT AAG TTC TGC 3´; 18 mer;
The primer pair amplifies a 516-bp cDNA fragment of the LIF gene.[17,34,46]
IL-6 S = 5´ATG AAC TCC TTC TCC ACA AGC GC 3´; 23 mer;
IL-6 A = 5´GAA GAG CCC TCA GGC TGG ACT G 3´; 22 mer;
The primer pair amplifies a 628-bp cDNA fragment of the IL-6 gene spanning four introns.[24,38]
gp130 S = 5´AAT GGG CAA CAC ACA AG 3´; 17 mer;
gp130 A = 5´AGT CAC AGG CAG GGA TAG 3´; 17 mer;
The primer pair amplifies a 774-bp cDNA fragment of the gp130-R gene.[16]
LIF-R S = 5´ATA CAG ATG GTG GAG TGG 3´; 18 mer;
LIF-R S = 5´TGA TGG GTG GAC AAT AGG 3´; 18 mer;
The primer pair amplifies a 420-bp cDNA fragment of the LIF-R gene spanning two introns.[18]
IL-6-R S = 5´CAT TGC CAT TGT TCT GAG GTT C 3´; 22 mer;
IL-6-R S = 5´AGT AGT CTG TAT TGC TGA TGT C 3´; 22 mer;
The primer pair amplifies a 251-bp cDNA fragment of the IL-6-R gene.[69]
Polymerase Chain Reaction
Ten µl of the diluted RT reaction mixture was used in a final volume of 100 µl containing PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.01% w/v gelatin), 50 pmol of each forward and reverse primer, 200 mM of each of the dNTPs, 0.5 U of Taq polymerase, and H2O. Samples were overlayed with 60 µl of mineral oil and amplified in 33 cycles: denaturing 40 seconds at 94šC, annealing 40 seconds using appropiate temperatures for the annealing step (at 59šC for OSM1, 57šC for OSM2, 58šC for LIF1, 56šC for LIF2, 60šC for IL-6, 51šC for gp130, 50šC for LIF-R, and 60šC for IL-6-R) and extension for 150 seconds at 72šC in a thermal cycler. Amplification was completed by a final incubation at 72šC for 10 minutes and stored at 4šC until further analysis.
To minimize cross contamination, positive displacements pipettes were used throughout the procedures and negative controls were performed for each assay. These data are not shown in the figures.
Agarose Gel Analysis
The PCR products were run on a 1.5% agarose gel in TAE buffer (40 mM Tris, 20 mM acetate, 2 mM EDTA pH 7.0) containing ethidium bromide (0.25 µg/ml) in the presence of a standard DNA size marker and under UV light. Amplified DNA fragments were cut out and the PCR products were extracted with the gel extraction kit. The final yields were each dissolved in 20 µl low TE, pH 8.
Direct Sequencing of the PCR Product
To obtain single-stranded cDNAs, the purified PCR products were digested with exonuclease III for 30 minutes at 37šC. The reactions were terminated at 80šC for 10 minutes. The reaction mixtures containing the single-stranded cDNAs were purified with the QIAquick spin-PCR purification kit. These purified fragments were sequenced by the dideoxynucleotide chain termination method,[52] with the Sequenase V.2.0 sequencing kit according to the manufacturer's instructions.
Cloning of PCR Products
Purified amplification products were end-repaired with the Klenow fragment DNA polymerase I according to the manufacturer's protocol. After heat inactivation, a kinase reaction was performed to ensure phosphorylated 5´-ends. The prepared DNA fragments were subsequently cloned into the multiple cloning site of the pUC18 plasmid. After ligation with T4 DNA ligase, an aliquot was transformed into the Escherichia coli strain Sure. Positive clones were selected by blue-white screening under selective conditions (100 µg/ml ampicillin) and verified by sequence analysis using standard primers for pUC18 derivates.
Sources of Supplies and Equipment
Antibodies against LIF, IL-6, and OSM and ELISAs were obtained from Biermann, Bad Homburg, Germany. Whatmann nitrocellulose filters were obtained from Bender and Hobein, Munich, Germany. The scintillation liquid was purchased from Canberra-Packard, Frankfurt, Germany. The Beta counter (model LS 1801) was manufactured by Beckman, Frankfurt, Germany. The Pepsin-HCl, ethidium bromide, and ribonuclease were supplied by Serva, Inc., Heidelberg, Germany. The mithramycin was obtained from Sigma, Deisenhofen, Germany. The cytometer (model ICP-22) was manufactured by Phywe, Gottingen, Germany; and the cytoanalyzer (model IN-96) was manufactured by Intertechnique, Mainz, Germany. The Centricon 10 columns were produced by Amicon, Witten, Germany. The rabbit anti--mouse antibody was supplied by Dako, Hamburg, Germany. The chromogen, standard DNA size marker, and T4 DNA ligase were supplied by Boehringer, Mannheim, Mannheim, Germany. The QuickPrep purification kit, Gene Quant spectrophotometer, and RNase-free DNase I were obtained from Pharmacia, Freiburg, Germany. The human placenta DNA, RNasin, and avian myeloblastosis virus-RT were supplied by Promega, Hilden, Germany. The Gen Zentrum HPLC was manufactured by MPI, Martinsried, Germany. The Taq polymerase was produced by Perkin Elmer Cetus, Heidelberg, Germany. The Quiagen gel extraction kit and QIAquick spin-PCR purification kit were obtained from Diagen, Heidelberg, Germany. The Sequenase sequencing kit came from Amergham Life Sciences, Brabinschweig, Germany. The polynucleotide kinase, primers for pUC18, and the plasmid pUC18 were supplied by New England Biolabs, Schwalbach, Germany. The Escherichia coli strain Sure was supplied by Stratagene, Heidelberg, Germany.
RESULTS
Reverse TranscriptionPolymerase Chain Reaction Experiments
Transcription of the cytokine genes LIF, IL-6, and OSM, their cognate receptors genes, and the STAT factors genes were examined by RT-PCR experiments. As shown in Fig. 1, all mRNA species can be detected in these assays, whereas negative controls remained negative (not shown). The 10 RT-PCR experiments for cultured meningioma cells produced evidence that all cytokines, their cognate receptors, and important factors of the downstream signaling cascade were actively transcribed in these cells. The RT-PCR products revealed the predicted size of 516 bp for LIF, of 628 bp for IL-6, and of 282 bp for OSM. As anticipated, the presence of mRNA's for LIF-R, IL-6-R, and gp130 was demonstrated in all cells (420 bp, 251 bp, 774 bp; Fig. 1). The sizes of the transcription factors STAT 1, 3, and 5a were 394 bp, 519 bp, and 529 bp, respectively (Fig. 1). All amplimers were found in each individual meningioma tested. All RT-PCR experiments were confirmed by cloning the amplification products and subsequently subjecting them to sequence analysis. All amplimers were identical compared with the published sequences.
Fig. 1. Western blot analyses. Upper left: Autoradiograms showing RT-PCR amplification product of the LIF and LIF-R genes. The RT-PCR products (cDNA) revealed the predicted sizes of 516 bp and 420 bp, respectively. Upper right: Autoradiograms showing RT-PCR amplification product of the IL-6 and IL-6-R genes. The RT-PCR products (cDNA) revealed the predicted sizes of 628 bp and 420 bp, respectively. Lower left: Autoradiograms revealing RT-PCR amplification product of the OSM and gp130 genes. The RT-PCR products (cDNA) revealed the predicted sizes of 282 bp and 774 bp, respectively. Lower right: Autoradiograms showing RT-PCR amplification products of STAT 1, 2, and 5 genes (signal transducers and activators of transcription). The RT-PCR products (cDNA) predicted the expected sizes of 394 bp, 591 bp, and 529 bp, respectively. Each amplification product was generated from the cDNA of cultured meningioma cells obtained in five patients. The results obtained in three patients (M1, M2, and M3) are presented. The set of amplification products are generated from the same cDNA.
Production of Cytokines Revealed by ELISA and Western Blot Experiments
The presence of secreted LIF, IL-6, and OSM proteins were demonstrated by ELISA of low passage meningioma cells (five experiments). The concentrations of secreted LIF, IL-6, and OSM proteins were estimated from 7.4 to 83 pg, 0.25 to 10.3 ng, and 29.8 to 829 fg per 106 cells at 24 hours, respectively (Table 1). During a culture period of 9 days, the amount of secreted LIF, IL-6, and OSM accumulated with increasing cell density in each individual experiment. However, no correlation between cell density and the amount of cytokine secretion could be drawn. To exclude the possibility that LIF, IL-6, or OSM had been artificially transferred to the culture medium by FCS, a 10-fold concentration of FCS was tested for LIF, IL-6, and OSM by ELISA. None of the serum samples reacted positively for the cytokines tested.
To evaluate the molecular size fraction of these cytokines and to prove that the secreted cytokines revealed the expected glycosylated forms, Western blot experiments were performed. The cytokines, LIF and IL-6, were secreted by cultured meningioma cells and revealed the expected size of 48 kD for LIF and 30 kD for IL-6 (Fig. 2). The amount of OSM secreted was too low to be detected by Western blot techniques.
Fig. 2. Western blots revealing a 48-kD glycosylated form of LIF (left) and a 30-kD glycosylated form of IL-6 (right). M1 and M2 = meningotheliomatous and fibromatous meningioma. C = control (recombinant nonglycosylated forms of LIF or IL-6). The blots were generated from conditioned medium of cultured meningioma cells.
Effects of rLIF, rIL-6, and rOSM on Meningioma Growth Rates as Determined by Cell Count, [3H]Thymidine Uptake, and DNA Flow Cytometry
Cell Count. To evaluate the role of these cytokines on meningioma cell growth, we performed cell culture experiments with meningioma tissue. Recombinant cytokines (rLIF, rIL-6, or rOSM) were tested separately in increasing concentrations of 1 ng to 100 ng/ml in culture medium containing 5% FCS, heat-inactivated medium, or ultraculture medium for 5 to 9 days. Recombinant LIF and rIL-6 did not alter the cell density significantly (Fig. 3b). Using monoclonal antibodies against endogenous LIF, IL-6, or IL-6-R, a similar biologically insignificant decrease was found in two-thirds of all meningiomas, whereas in one meningioma no effect was observed (data not shown). In contrast, rOSM was found to be a potent inhibitor of meningioma cell proliferation. In culture medium containing 5% FCS (10 experiments), rOSM acted dose dependently and reduced meningioma cell growth to 34% compared with control experiments (Figs. 3b and 4a).
Fig. 3. Left: Graph illustrating the effect (given as a percentage) of 10 ng/ml rIL-6 on [3H]thymidine uptake (relative to control) in meningioma cultures over a period of 30 hours. The [3H]thymidine incorporation was decreased at 6 hours, increased at 12 hours, and adjusted to control levels at 18, 24, and 30 hours. These data indicate that [3H]thymidine uptake in the early period did not reflect any effects of cell proliferation after IL-6 administration to cultured meningioma cells. Under the same conditions rOSM (10 ng/ml) persistently decreased the [3H]thymidine incorporation at 12 to 30 hours to 62%. Each point represents the average of three cultures (>105 cells/flask) ± its standard deviation (SD). Absolute counts are between 104 and 2 X 105 cpm/flask (* = p < 0.05 vs. control; ** = p < 0.01 vs. control). This experiment (one example of five) was performed in 5% FCS with medium and drug change every 6 hours. Right: Graph illustrating the effects (given as a percentage) of rLIF, rIL-6, and rOSM after a 7-day incubation period on cell number (relative to control) in meningioma cultures in various concentrations. These experiments were performed in parallel with the same stock of cells. No change in cell number after rLIF or rIL-6 treatment was observed, whereas rOSM inhibited cell growth dose dependently down to 34%. Each point represents the average of three cultures (gt105 cells/flask) ± SD. Data are from one of five similar experiments.
[3H]Thymidine Uptake. There were many effects on rIL-6 by [3H]thymidine incorporation in 5% FCS in which medium was changed every 6 hours over a period of 30 hours (five experiments). The [3H]thymidine incorporation varied significantly: there was a decrease to 62% at 6 hours; an increase to 148% at 12 hours; and an adjustment to the control cultures at 18, 24, and 30 hours (Fig. 3a). When the total cell number was determined at the same time points in a parallel experiments with the same stock of cells, no increase within the first 30 hours was observed. In contrast, rOSM significantly lowered the [3H]thymidine uptake to 63% after between 12 and 30 hours of incubation compared with control experiments (Fig. 3a).
Fig. 4. Upper: Histogram showing cell density measurements after a 7-day incubation period with rOSM. Recombinant Oncostatin M decreased growth (cell density) of human meningioma cultures dose dependently. The maximum decrease was 50% and 70% at dosages of 50 and 100 ng/ml, respectively. Bar "A" represents the number of seeded cells; bar "B" represents the number of cells after 48 hours (lag phase) before treatment with rOSM; bar "C" represents the number of cells (controls) at the end of the experiment. Each bar represents the means ± SDs of experiments performed in triplicate with meningioma cell cultures (** = p < 0.01 vs. control; *** = p < 0.001 vs. control). Lower: Results of DNA flow cytometry showing a dose-dependent effect of rOSM on cell cycle phases after a 7-day incubation period, using cell cultures derived from the same meningioma used in the experiments shown in Fig. 4 upper (WHO Grade I meningotheliomatous meningioma). Recombinant OSM at concentrations of 10 ng/ml, 50 ng/ml, or 100 ng/ml significantly increased the percentage of cells in the G2/M phase to 7%, 10%, and 14%, respectively; (p < 0.01, p < 0.001, and p < 0.001 vs. control 1.7%). C = control culture; rOSM 10, 50, and 100 ng/ml = treated cultures. Results are given in percentage of DNA in cell cycle phases. Data represent means ± SDs of experiments performed in triplicate. Statistical significance: ** = p < 0.01, *** = p < 0.001, and *** = p < 0.001, respectively.
DNA-Flow Cytometry. Effects on growth rate were confirmed by DNA flow cytometric analysis. Recombinant OSM changed the cell cycle phases significantly in concentrations of 10 ng/ml, 50 ng/ml, and 100 ng/ml (rOSM), whereas rLIF or rIL-6 was without effect. Growth rates were determined in parallel with the individual stock of meningioma cells. Five experiments were performed on cell cultures derived from three WHO Grade I and two WHO Grade II meningotheliomatous meningiomas. Recombinant OSM (10 ng/ml) significantly increased the percentage of cells in the G2/M phase to 7.1% (compared with control 1.7%; p < 0.001) (Fig. 4b). In cells to which 50 ng/ml and 100 ng/ml of rOSM had been added, this effect was more pronounced; rOSM increased the G2/M phase to 10.3% and 14.3%, respectively (compared with control 1.7%; p < 0.001, both) (Fig. 4b).
In summary, rLIF and rIL-6 have no significant effects on meningioma cell growth, whereas rOSM strongly inhibited meningioma cell proliferation as demonstrated by cell count, [3H]thymidine uptake, and cell cycle analysis.
DISCUSSION
Cytokines have been shown to be important to disease processes such as cancer and intracranial tumors (for example glioblastoma multiforme and meningiomas).[60,64] Transfection experiments with genes for IL-4 and IL-7 impede tumor growth,[3,70] whereas transforming growth factor-ß and IL-10 may support tumor development by suppressing antitumoral immune responses.[12,25,43,73] Recently IL-6 and IL-1ß, which have been suggested to play a synergistic role in gliomas,[32,64] were shown to be secreted in vitro by cultured meningioma cells themselves, rather than by contaminated lymphocytes as determined by fluorescent automated cell sorting analysis.[32] The ability to manipulate cytokine secretion in meningiomas and, thus growth inhibition as well, may provide an impetus for further progress in this field.[43]
We have investigated the expression of LIF, IL-6, and OSM, which belong to a family of cytokines that uses a similar signaling pathway and a common signal transducer molecule called gp130. On the protein level, LIF and OSM share a 30% homology,[50] whereas the homology to IL-6 is low.[5] In the case of OSM and LIF, signaling is mediated by the heterodimerization of one signal transducer molecule, gp130, and one specific ligand-binding domain (LIF-R and OSM-R). In the case of IL-6, the homodimerization of two gp130-R and two IL-6-R molecules is required for biological activity.[13,16,18,62] The gp130-mediated signal pathway involves JAK1, JAK2, and TYK2 as specific protein tyrosine kinases mediating phosphorylation to a cell type-specific set of STAT factors (signal transducer and activator of transcription).[1,49,67] Although a redundant signaling mechanism was demonstrated, pleiotropic biological effects were observed in different cells and tissues.[16,18,28,50]
The present study clearly demonstrates that all three cytokines, their related receptors, and specific STAT factors were actively transcribed in meningioma cells (Fig. 1). In addition, the secretion of LIF, IL-6, and OSM from cultured meningioma cells was demonstrated by ELISA and Western blot experiments. These data indicate an autocrine loop for LIF, IL-6, and OSM in meningioma cells.
To investigate the biological effects of all three cytokines, distinct amounts of rLIF, rIL-6, and rOSM were added to growing meningioma cells. In long-term cultures (79 days), rOSM showed a strong inhibitory effect on meningioma cell proliferation. Using rLIF and rIL-6, only weak or no growth-modulatory effects were observed. To exclude the possibility that the observed weak effects of rLIF and rIL-6 on meningioma cell growth were disguised by endogenous LIF and IL-6 production, an additional set of experiments was performed using neutralizing antibodies against LIF, IL-6, and IL-6-R molecules. However, no biologically relevant growth-promoting or inhibiting activity on meningioma cells was observed in vitro.
To study these weak effects in more detail, [3H]thymidine incorporation experiments with rIL-6 were performed. In experiments in which the media were changed every 6 hours over a period of 30 hours, a decrease of tritium uptake into the genomic DNA was observed after 6 hours and an increase after 12 hours. The cell density of treated and untreated cultures determined at the same time points was not altered by rIL-6. The fact that the cell density was not altered indicates that the [3H]thymidine incorporation during early periods does not reflect any growth-promoting or inhibiting effects of rIL-6 on meningioma cells; however, it may indicate a yet unknown function in the cell cycle. The observed effects may be explained by an accelerated G1/S phase and an arrest of cells in G0 phase. No increase in cell number can be detected, but the increase and delay for tritium incorporation can be explained. This is in contrast to recently published data that indicated that IL-6 might mediate autocrine growthinhibitory effects on meningioma cells.[63] With respect to IL-6, our data are in agreement with Boyle-Walsh, et al.,[7] who demonstrated that rIL-6 alone has either no effect or only a moderate proliferative effect. We conclude that the mitogenic effect of rLIF and rIL-6 on cultured meningioma cells is of minor biological relevance.
Only rOSM showed a significant inhibitory effect on meningioma cell growth as demonstrated by cell count, [3H]thymidine incorporation, and cell cycle analysis. In long-term culture experiments over a period of 7 to 9 days, the absolute cell number dropped to 34% compared with control experiments. The observed effect was obtained in different experiments, thus indicating that rOSM is a potent inhibitor for meningioma cell growth in vitro. The OSM-R represents a potential target for the antimitotic action of rOSM. Therefore, rOSM could be a candidate for adjuvant medical treatment in patients in whom surgical treatment has failed.
Acknowledgment
The authors thank Prof. Dr. P. Thierauf, Department of Pathology, Neuropathology Division, University of Erlangen, for histological classification of the meningiomas.
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Manuscript received February 17, 1997.
Accepted in final form March 17, 1997.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, Schr 341/7-2) and by the Johannes and Frieda Marohn Stiftung (SCHRE/92).
Primers for transcription factors (STAT 1, STAT 3, and STAT 5) are a gift from Dr. J. Ripperger, Department of Genetics, University of Erlangen-Nürnberg.[49]
Address reprint requests to: Uwe M. H. Schrell, M.D., Neurochirurgische Klinik, der Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany.
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