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* Department of Surgery, ABSTRACT L-Glutamine is the most abundant free amino acid of the human body and is essential for the culture of many cell types. Clinically, reduction of glutamine by administration of glutaminase or the use of glutamine analogs is a common therapy for patients with acute lymphocytic leukemia. In the current study, we investigated the influence of glutamine concentrations on the human myelomonocytic cell line U937. Decreasing the glutamine concentration evoked a reduction in DNA synthesis (R2 = 0.9885, P < 0.0001), increased cell volume (P < 0.01) and the cytoplasm/nuclear ratio, and enhanced the development of vacuoles but did not influence cell viability. Culturing cells in reduced concentrations of glutamine augmented the percentage of cells expressing CD64 (Fc receptor for IgG/Fc INTRODUCTION Glutamine is the most abundant amino acid in the body and is important in the growth of normal and neoplastic cells (Kovacevic and Morris 1972 We previously demonstrated that administration of asparaginase from Escherichia coli to patients with acute leukemia evokes an acute plasma glutamine reduction, because the asparaginase was contaminated with glutaminase (Ollenschläger et al. 1988 The effect of glutamine on the metabolic and immunological behavior of cells has been investigated in several systems. Glutamine utilization is essential for lymphocyte proliferation, influences differentiation of B cells into plasma cells and stimulates the spontaneous transformation of NIH-3T3 cells (Calder 1994 The established human cell line U937, with monoblastic and immature monocytic characteristics (Sundström and Nillson 1976), can be induced to differentiate into monocyte/macrophage-like cells with various stimuli, including 12-O-tetradecanoylphorbol-13-acetate (Nilsson et al. 1980 MATERIALS AND METHODS Institute of General and Experimental Pathology and Institute of Clinical Pathology, University of Vienna, 1090 Vienna, Austria
RI, P < 0.01), CD11b (complement receptor type 3/CR3, P < 0.001) and CD71 (transferrin receptor, P < 0.05). The percentage of U937 cells expressing CD23 (low affinity receptor for IgE/Fc
RII) was increased at low concentrations of glutamine at both the protein (P < 0.01) and mRNA levels. The percentage of U937 cells phagocytizing opsonized E. coli (P < 0.001) or latex particles (P < 0.001) was enhanced by lowering the glutamine concentration. In conclusion, reducing glutamine concentration causes differentiation of the cell line U937 along the monocytic pathway. These effects may indicate a mechanistic basis for prior published evidence that glutaminase and glutamine antagonists are effective anti-tumor agents.
, Roth et al. 1988b) as well as for many types of cultured cells (Eagle 1959, Roth et al. 1996). A glutamine reduction, obtained by the application of a glutaminase-asparaginase enzyme cocktail, has been found to be a biochemically rational therapy for the treatment of patients with acute leukemia or intra-abdominal metastatic cancers (Holcenberg et al. 1983, Spiers and Wade 1976). Similarly, the administration of antagonists of glutamine such as azaserine, 6-diazo-5-oxo-L-norleucine, acivicin and azotomycin evokes significant anti-tumor effects (Catane et al. 1979).
). One hour after asparaginase-glutaminase application, plasma glutamine concentrations decreased to 5% of initial values, and during 14 d of drug application plasma glutamine levels in fasting subjects fell to a median of 63% of pre-therapeutic levels. This glutamine reduction may be partially responsible for the anti-tumor effect of asparaginase-glutaminase but may also have deleterious effects on liver function and on the immune system.
, Crawford and Cohen 1985, Rubin 1990). A glutamine-dependent modulation of phenotype and function has also been described in human colon cancer cell lines (Turowski et al. 1994). Recently we showed that lowering the glutamine concentration in culture medium from 2 to 0.2 mmol/L reduces the expression of HLA-DR on human monocytes, decreases tetanus toxoid-induced antigen presentation and influences the expression of several cell surface antigens (Spittler et al. 1995).
), 9-cis retinoic acid and 1
,25-dihydroxycholecalciferol (Amento et al. 1984, Botling et al. 1996, Olsson and Breitman 1982, Spittler et al. 1997), interferon- (IFN-)4 and tumor necrosis factor- (TNF-) (Harris et al. 1985, Trinchieri et al. 1986). Cell differentiation, proliferation and activation are accompanied by changes in morphology and the modulation of several antigens. Maturation of monocytes along the monocytic pathway includes reduced DNA synthesis, development of phagocytic activity and enhanced expression of the receptors for immunoglobulin G (FcR) and complement components (CR). Therefore in the present study we investigated the action of glutamine reduction on the capacity of the myelomonocytic cell line U937 to proliferate and synthesize DNA, to express various cell surface molecules, and to phagocytize opsonized E. coli or latex particles.
) was initially obtained from the American Type Culture Collection (Rockville, MD). U937 cells were cultured at an initial density of 3 × 105 cells/mL in phenol red-free RPMI 1640 medium (BioWhittaker, Walkesville, MD) supplemented with 1 × 105 U/L penicillin, 1 mg/L streptomycin, 2 mmol/L glutamine (all from Flow Laboratories, Irvine, Scotland, U.K.) and 100 mL/L heat-inactivated fetal calf serum (FCS) (Gibco, Paisley, U.K.). This culture medium was additionally supplemented with various concentrations of glutamine (0.05, 0.1, 0.2, 0.3, 0.6, 1, 2 mmol/L) freshly prepared from a stock solution obtained from Flow Laboratories and stored at 
20°C. Because FCS contained 0.540 mmol/L glutamine, the lowest concentration of glutamine that could be achieved in culture medium was approximately 0.05 mmol/L. Control experiments were performed with ornithine -ketoglutarate (OKG) (Sigma Chemical, St. Louis, MO), which is a salt formed of one molecule of -ketoglutarate and two molecules of ornithine. Ornithine -ketoglutarate was used at concentrations of 0, 0.1, 0.2, 0.3, 0.6, 1 and 2 mmol/L. Concentrations of glutamine in cell culture medium were measured using HPLC (Roth et al. 1988a). The U937 cells were cultured in six-well plates (Costar Corporation, Cambridge, MA) for 4 d at a density of 1.5 × 106 cells/5 mL in a fully humidified air atmosphere containing 5% CO2 at 37°C.
).
). Quantitative measurements of the frequency distribution (
1 × 104 events) was analyzed with Multicycle AV 3.0 software (Phoenix Flow Systems, San Diego, CA).
. [3H]Inulin was used as marker for extracellular space and [14C]urea was used as marker for intra- and extracellular space (vom-Dahl et al. 1991). U937 cells (2.5 × 107) were incubated in 2.5 mL of the above-mentioned medium for the indicated period of time, and then 3.7 kBq of [3H]inulin and 1.85 kBq of [14C]urea were added to the incubations. After 10 min, the cell suspension was gently centrifuged (170 × g) for 5 min, and 3H and 14C radioactivity was measured in the supernatant and the pellet by 
liquid scintillation, respectively. The intracellular water space was calculated from the amount of 14C found in the pellet after correction for residual extracellular water in the pellet by using the specific radioactivities in the supernatant (Oehler et al. 1996).
Monoclonal antibodies.
All monoclonal antibodies (MoAb) were either conjugated with fluoresceinisothiocyanate (FITC) or phycoerythrin (PE). Monoclonal antibodies anti-FcRI/CD64 (clone 32.2, FITC) and anti-FcRII/CD32 (clone IV.3, FITC) were obtained from Medarex Inc. (West Lebanon, NH); Leu-20 (anti-FcRII/CD23; clone EBVCS-5, PE), Leu-11a (anti-FcRIII/CD16; clone NKP15, FITC), Leu-15 (anti-CR3/CD11b; clone D12, PE), Leu-M5 (anti-CR4/CD11c; clone S-HCL-3, PE), Leu-M7 (anti-CD13; clone L138, PE) and anti-HLA-DR (clone L243, FITC) were products of Becton Dickinson; anti-CD14 (clone TÜK4, FITC) and anti-transferrin receptor/CD71 (clone Ber-T9, FITC) were obtained from DAKO (Glostrup, Denmark); anti-CR1/CD35 (clone J3.D3, FITC), anti-CD54/ICAM-1 (clone 84H10, FITC), anti-CD33 (clone D3HL60.251, FITC), anti-CD11a (clone 25.3.1, FITC), mouse isotype controls (IgG1, clone 679.1Mc7, FITC or PE, and IgG2a, clone U7.27, FITC or PE) were obtained from Immunotech (Marseille, France). Anti-HLA-ABC (clone W6/32-HL, FITC) was purchased from Cymbus Science Limited (Southampton, U.K.).
Immunofluorescence and flow cytometry.
Cells were harvested, washed and resuspended in phenol red-free Hanks' balanced salt solution (HBSS) (BioWhittaker, Walkesville, MD) containing 3 g/L bovine serum albumin (U.S. Biochemical, Cleveland, OH) and 1 g/L NaN3. After preincubation of 5 × 105 cells for 30 min at 4°C with 200 mL/L human serum of blood group A and B (Biotest, Dreieich, Germany) dissolved in HBSS containing 3 g/L bovine serum albumin and 1 g/L NaN3, cells were washed twice in HBSS. Monoclonal antibody concentrations for staining 1 × 106 cells as given by the manufacturers were diluted in HBSS supplemented with 100 mL/L human AB serum to a volume of 50 µL. Monoclonal antibodies and 5 × 105 cells were incubated for another 30 min at 4°C, washed three times and resuspended in 300 µL of HBSS. Finally 50 mg/L of propidium iodide (Sigma Chemical) was added to cells stained with a FITC-conjugated MoAb to exclude dead cells (Spittler et al. 1995). At least 1 × 104 cells were analyzed in a FACScan (Becton Dickinson). The data were collected with four-decade logarithmic amplification and expressed as a percentage of stained cells. During this study, identical instrument settings were used.
Phagocytosis.
The phagocytic capacity of U937 cells was measured by incubating cells (2 × 105) with FITC-labeled opsonized E. coli (2 × 108; Operators Manual Phagotest®, Orpegen Pharma, Heidelberg, Germany) for 1 h at 37°C (time established in preliminary experiments). Subsequently cells were washed three times with HBSS and analyzed by FACS (Spittler et al. 1995). Fluorescein isothiocyanate-labeled latex beads (Fluoresbrite plain) with a diameter of 0.75 µm were obtained from Polysciences Inc. (Warrington, PA). U937 cells (2 × 105) and beads (8 × 107) were incubated for 1 h at 37°C (Sundström and Nilsson 1976), layered on FCS, centrifuged and washed. The percentage of phagocytic cells was determined by FACScan analysis.
RNA isolation and Northern blot analysis.
Total RNA was prepared from 1 × 107 U937 cells using the guanidine isothiocyanate method (Chomczynski and Sachi 1987). RNA was denaturated in 1 mol/L glyoxal, 2.5 mmol/L sodium phosphate (pH 6.8), 1.25 mmol/L EDTA, 320 g/L dimethylsulfoxide at 50°C for 40 min. Following electrophoresis in 15 g/L agarose gels, RNA was blotted onto Hybond-N nylon membranes (Amersham, Little Chalfont, U.K.) in 20× SSC (1× SSC is 0.15 mol/L NaCl, 0.015 mol/L sodium citrate, pH 7.0). After crosslinking with UV, blots were subjected to prehybridization for 4 h at 42°C in 500 g/L formamide, 5× SSPE (20× SSPE is 3 mol/L NaCl, 20 mmol/L EDTA, 0.3 mol/L sodium phosphate, pH 7.4), 5× Denhards (50× Denhards is 10 g/L Ficoll 400, 10 g/L polyvinylpyrrolidone, 10 g/L bovine serum albumin), 5 g/L SDS, 100 mg/L salmon sperm DNA containing 25.9 kBq of denatured [32P]dCTP (Amersham)-labeled random primed DNA probes. The probes used for hybridization were PCR products using U937 cell cDNA as template and specific primers for CD23 (5
-CCC ACC AGC CGG AGC CAG-3/5-CTG GGA AGG CAG GGG CCA-3, with an annealing temperature of 65°C and a product length of 416 bp) and beta-actin (5-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3/5CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3, with an annealing temperature of 60°C and a product length of 838 bp). To remove unbound probes after hybridization, membranes were washed three times with 2× SSC, 1 g/L SDS for 10 min, twice with 25 mmol/L sodium phosphate (pH 7.2), 1 g/L SDS, 1 mmol/L EDTA and twice with 25 mmol/L sodium phosphate (pH 7.2), 10 g/L SDS, 1 mmol/L EDTA for 20 min at 53°C. To visualize the bound probes, filters were exposed to X-ray films (AR X-OMAT X-ray film; Kodak, Vienna, Austria) at 70°C with intensifying screens. By exposures of different lengths of time we defined the linear range of the film. The linear range of the film was estimated and scanned using a densitometer (PDI, Pharmacia-Biotech, Upsala, Sweden).
Statistical analysis.
Data were analyzed with a statistical software package (SYSTAT 5.01 for Windows®). To establish the dose-response relationships, a regression analysis of the dose vs. the [3H]thymidine uptake using the MGLH procedure was performed. To estimate the relationship of the percentage of CD23+ cells, glutamine concentrations and time of incubation, a multiple regression was calculated (Hiesmayr et al. 1996). The goodness of fit was determined by the R2 value. Student's t test was used for all pairwise comparisons. Data are presented as means ± SD. A P value <0.05 was considered statistically significant.
RESULTS
-ketoglutarate on cell proliferation.
U937 cells were cultured for 4 d with different concentrations of glutamine or OKG. Glutamine reduction diminished the proliferation of U937 cells in a dose-related manner (R2 = 0.9885, Fig. 1). The maximum effect was observed at a glutamine concentration of 0.05 mmol/L, at which [3H]thymidine uptake was reduced by up to 70% compared with cells cultured in 2 mmol/L glutamine. In control experiments, to study whether glutamine may be replaced by other amino acids for energy source, U937 cells were cultured with OKG. As shown further in Figure 1, [3H]thymidine uptake was not influenced by increasing concentrations of OKG (R2 = 0.0185) from 0.05 to 2 mmol/L. Furthermore, the reduced [3H]thymidine uptake during OKG culture was similar to that for culture in 0.05 mmol/L glutamine.
-ketoglutarate (OKG) concentrations on U937 cell proliferation. To determine DNA synthesis, cells were cultured for 4 d with the indicated concentrations of glutamine or OKG at 37°C and pulsed for 6 h with [3H]thymidine (37 kBq/well). Subsequently, cells were harvested onto glass fiber filters with a semiautomatic cell harvester and dried. The incorporated activity was measured by -counter liquid scintillation. Data from five experiments are presented as means ± SD. To establish the dose-response relationship, a regression analysis of the dose vs. the [3H]thymidine uptake using the MGLH procedure was calculated.
1 × 104 events) was performed with the Multicycle AV 3.0 software. Histograms are from one representative experiment of three.
Fig. 3.
Influence of glutamine on morphological features of U937 cells. Cells were cultured for 4 d in tissue culture medium containing 0.05 mmol/L glutamine (panel a) or 2 mmol/L glutamine (panel b). Cells were then harvested, pelleted onto glass slides using a cytospin centrifuge and subsequently stained with hematoxylin and eosin. Cells cultured in 0.05 mmol/L glutamine (panel a) were larger, contained more vacuoles (black arrows), showed increased cytoplasm and a greater cytoplasm/nucleus ratio and fewer mitotic figures (white arrows) than cells cultured in 2 mmol/L glutamine (panel b). All these features suggest differentiation of U937 cells cultured with 0.05 mmol/L glutamine.
[View Larger Version of this Image (140K GIF file)]
Table 1.
Influence of glutamine (Gln) and ornithine 
Fig. 4.
Influence of glutamine on size of cultured U937 cells. Cells were cultured for 4 d with 2.0 or 0.05 mmol/L glutamine. Cell volume was determined using [3H]inulin and [14C]urea. The intracellular water space was calculated from the 14C radioactivity found in the pellet after correction for residual extracellular water in the pellet ([3H]inulin) by using the specific radioactivities in the supernatant as described in Materials and Methods. Each value is the mean ± SD of four experiments. **Significantly different from 2.0 mmol/L glutamine (P < 0.01).
[View Larger Version of this Image (35K GIF file)]
-ketoglutarate on cell surface antigen expression.
Cells were cultured for 4 d with different concentrations of glutamine, harvested and stained with a panel of MoAb. The percentage of U937 cells expressing constitutively cell surface markers such as FcRII/CD23, FcRI/CD64, CR3/CD11b and the receptor for transferrin/CD71 was significantly greater for cells cultured in 0.05 mmol/L glutamine rather than in 2 mmol/L glutamine (Table 1). The expression of FcRII/CD32, CR4/CD11c, CD11a, CR1/CD35, HLA-ABC, CD13 and CD54 was not significantly enhanced on U937 cells cultured with 0.05 mmol/L glutamine (Table 1). Antigens that were not constitutively expressed on U937 cells, such as FcRIII/CD16, CD14 and HLA-DR, were not induced by 0.05 mmol/L glutamine (Table 1). Interestingly, the percentage of cells expressing CD23, CD64, CD11b, CD71 and CD54 antigens was similar on U937 cells cultured in 2 mmol/L OKG or 0.05 mmol/L glutamine (see Table 1).
-ketoglutarate (OKG) on the phenotype of U937 cells1
DISCUSSION RII-specific mRNA was induced by interleukin (IL)-4 (1 × 105 U/L), an effect similar to that obtained with 0.05 mmol/L glutamine. Furthermore, the IL-4-triggered mRNA expression could be enhanced by incubating the cells in 0.05 mmol/L glutamine.
Fig. 5.
Time course and dose dependency of glutamine concentration on CD23 expression in cultured U937 cells. Cells were cultured for the indicated times with 0.05, 0.2, 0.6 and 2 mmol/L glutamine, harvested, washed and resuspended in phenol red-free Hanks' balanced salt solution (HBSS) containing 3 g/L bovine serum and 1 g/L NaN3. After preincubation of 5 × 105 cells for 30 min on ice with 20% human serum of blood group A and B dissolved in HBSS containing 3 g/L bovine serum albumin and 1 g/L NaN3, cells were washed in HBSS. Monoclonal antibody concentration for 1 × 106 cells as given by the manufacturer was diluted in HBSS to a volume of 50 µL. Monoclonal antibodies and 5 × 105 cells were incubated for another 30 min on ice. Cells were then washed three times and resuspended in 300 µL of HBSS. At least 1 × 104 cells were analyzed in a FACScan. The data were collected with four-decade logarithmic amplification and expressed as the percentage of CD23 antigen-bearing U937 cells. Data are from one representative experiment of three. The formula shows the results of the multiple regression analysis.
[View Larger Version of this Image (19K GIF file)]
Fig. 6.
Influence of glutamine on CD23-specific mRNA in cultured U937 cells. Total RNA was isolated from U937 cells cultured for 4 d in tissue culture medium containing 0.05 mmol/L glutamine (lane 1), 2 mmol/L glutamine (lane 2), 0.05 mmol/L glutamine and interleukin (IL)-4 (1 × 105 U/L; lane 3) or 2 mmol/L glutamine and IL-4 (1 × 105 U/L; lane 4). The blots were hybridized with oligonucleotides specific for CD23, or -actin to account for discrepancies in loading. To compare the quantities of CD23-specific mRNA in different samples, the CD23-specific signal was measured by densitometry and normalized to the -actin-specific signal. Cells cultured in 0.05 mmol/L glutamine showed higher level of CD23-specific mRNA as quantified by optical densitometry × mm (5.108, lane 1) than cells cultured in 2 mmol/L glutamine (3.954, lane 2). Cells cultured in 0.05 mmol/L glutamine and IL-4 showed showed higher levels of CD23-specific mRNA (8.331, lane 3) than cells cultured in 2 mmol/L glutamine and IL-4 (5.283, lane 4).
[View Larger Version of this Image (76K GIF file)]
Fig. 7.
Effects of glutamine on phagocytosis of E. coli or latex particles by cultured U937 cells. Cells were cultured for 4 d with the indicated concentrations of glutamine, harvested and washed twice. The U937 cells and fluorescein isothiocyanate (FITC)-conjugated E. coli or FITC-labeled latex beads were incubated for 1 h at 37°C as described in Materials and Methods. The percentage of phagocytic cells was determined by FACScan analysis. Hatched histograms indicate the data from the control group, obtained with U937 cells incubated with FITC-conjugated E. coli or latex beads for 1 h at 0°C, a procedure known to inhibit phagocytosis. Histograms are from one representative experiment. Furthermore, when the mean ± SD of four experiments was calculated, the percentage of U937 cells ingesting opsonized E. coli was significantly enhanced by lowering the glutamine concentration from 2 mmol/L (23.6 ± 6.6%) to 0.05 mmol/L (59.5 ± 7.2%, P < 0.001), as was the capacity to phagocytose FITC-labeled latex beads (26.1 ± 2.2% vs. 51.9 ± 5.5%, P < 0.001).
[View Larger Version of this Image (26K GIF file)]
RI/CD64, FcRII/CD23 and CR3/CD11b were significantly augmented, and the capacity to phagocytize opsonized E. coli as well as latex particles was markedly enhanced. These data strongly suggest that low concentrations of glutamine induced differentiation of U937 cells from the promonocyte into more mature and less proliferative monocyte-like cells.
). Phenylacetate causes a glutamine reduction by conjugating glutamine to phenylacetate, which is subsequently excreted in the urine. A glutamine dose-dependent modulation of phenotype and proliferation rate has been demonstrated in human colon carcinoma cell lines Caco-2 and SW620 (Turowski et al. 1994). Glutamine supplementation increases proliferation, decreases differentiation and diminishes adhesion matrix proteins associated with decreased integrin expression. Studies performed with the human HL-60 promyelocytic leukemia cells have shown that arginine deprivation induces alterations in cell morphology, nonspecific esterase activity, hydrogen peroxide production and expression of CD14 and CD11b on the cell surface (Nichols and Weinberg 1989). Subsequent experiments performed in the same laboratory revealed that acivicin, a glutamine anti-metabolite, decreased cell growth and caused differentiation of HL-60 cells along a monocytic pathway (Nichols et al. 1989). Furthermore, acivicin potentiated the known differentiating effects of IFN-, TNF-, dihydroxycholecalciferol, dimethylsulfoxide and retinoic acid. The effects of acivicin on cell growth and differentiation are possibly related to the fact that acivicin inhibits several glutamine-utilizing enzymes.
, IL-1, c-myc and c-myb in HL-60 cells (Weinberg et al. 1992). The increased production of TNF- and IL-1 was the result of induced cytokine-specific mRNA transcription. Our data on CD23 expression also indicate that reduced glutamine concentration stimulates surface antigen expression by increasing the percentage of CD23+ cells in a concentration- and time-dependent manner. Northern blot analysis showed that U937 cells contain CD23-specific mRNA and that its level was increased in cells cultured with 0.05 mmol/L glutamine. These data suggest that augmented surface CD23 expression is controlled at the gene level. A direct effect of glutamine on gene regulation has been shown for heat-shock proteins in Chinese hamster ovary cells (Cai et al. 1991), Drosophila Kc cells (Sanders and Kon 1991) and opossum kidney cells (Nissim et al. 1993). On the level of translation it was shown that administration of asparaginase-glutaminase inhibits mouse retroviral disease by stopping translation of mRNA at the gag-pol junction (Roberts and McGregor 1991).
). Because lowering glutamine concentrations in culture medium may reduce both nitrogen and energy availability to cells, in some experiments glutamine was replaced OKG. Our results (see Fig. 1 and Table 1) strongly suggest that changes in cell proliferation and cell phenotype are caused by reduced glutamine concentrations, and not influenced by alterations in nitrogen or energy content of the culture medium.
). Treatment of patients with myelodysplastic syndromes with agents that induce differentiation pushes immature progenitors out of the mitotic compartment into a nonmitotic stage and increases the number of functional hematopoietic cells (Spriggs et al. 1986). The response of leukemia cells to glutamine deprivation may partly be responsible for the anti-tumor effects of asparaginase-glutaminase therapy in patients with leukemia. Our study presents evidence that glutamine is an effective regulator of cell behavior influencing cell growth and differentiation and supports the clinical use of glutaminase, the major enzyme of glutamine hydrolysis.
FOOTNOTES
, interferon-; IL, interleukin; MCF, mean channel fluorescence; MoAb, monoclonal antibody; OKG, ornithine -ketoglutarate; PE, phycoerythrin; TNF-, tumor necrosis factor-.
Manuscript received 19 February 1997. Initial reviews completed 1 April 1997. Revision accepted 18 July 1997.
LITERATURE CITED
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ABSTRACT
