Regulation of the Proliferation of the Established Human Monoblast Cell Line, U937, at the Single Cell Level1

[CANCER RESEARCH 46, 3309-3312, July 1986] Regulation of the Proliferation of the Established Human Monoblast Cell Line, U937, at the Single Cell Level1

Ellin Berman, Berish Y. Rubin, and Hal E. Broxmeyer2

Department of Developmental Hematopoiesis, Memorial Sloan Kettering Cancer Center [E. B.J; Department of Lymphokine Biology, The Lindsley F. Kimball Research Institute of the New York Blood Center, New York, New York 10021 [B. Y. R.J; and Departments of Medicine, Hematology /Oncology, Microbiology and Immunology, Indiana University School of Medicine, Walther Medical Research Institute and the Elks Cancer Research Center, Indianapolis, Indiana 46223 Â¡H.E. B.]

ABSTRACT pliages and T-lymphocytes (4, 12). Colony formation by MHC class II antigen-positive U937 CFC can be suppressed by puri U937 cells, an established monoblast or early monocyte cell line, were fied iron-saturated LF and TF and this is associated with assessed as a model in vitro for the regulation of cell growth at the single induction of MHC class II antigens on U937 CFC by HuIFN- cell level. Colony formation by 500 U937 cells, preinduced to a state of â€¢y(13,14). Dialyzed U937 CM does not influence the cloning responsiveness to lactoferrin (LF) by incubation with human y Interferon was suppressed by LF. LF-suppressed colony formation was restored by efficiency of U937 cells but does restore colony formation partially purified growth activity derived from U937 cells. The release of suppressed by LF or TF without inactivating the LF or TF growth factor(s) into conditioned medium required concentrations of >51III (15). These results were similar to studies reported previously U937 cells/ml and this release was dependent on the length of time that with a murine myelomonocytic leukemia cell line, WEHI-3, the cells conditioned the culture medium. This release was suppressed that had been adapted to culture (16). The studies reported by LF. U937 cells were induced to a state of responsiveness to LF by herein utilize HuIFN-7, LF, TF and PP-U937-GA to present incubation with human 7 Interferon, washed, and plated as a single cell evidence for regulation of growth at the level of single isolated per well. Individual cells formed colonies with a cloning efficiency of U937 cells. -50% which equalled the cloning efficiency detected when 500 U937 cells/ml were plated, suggesting that U937 colony forming cells might contain endogenous growth activity. Detection of these endogenous MATERIALS AND METHODS growth activities required the use of LF. The cloning efficiency of Cell Line. U937 cells (2, 5) were grown in McCoy's 5A medium individually isolated U937 cells was suppressed by -50% with LF, supplemented with 10% heat-inactivated (56'C for 30 min) FBS (Hy- similar to the LF suppression of colony formation when 500 cells/ml were plated. That the LF-suppressed U937 colony forming cells required clone, Loga, UT), vitamins, sodium pyruvate, essential and nonessential amino acids, serine, L-glutamine, and asparagine (Gibco Laboratories, growth activity was suggested as the cloning efficiency of LF-suppressed Grand Island, NY) at 37Â°Cina humidified atmosphere of 5% CO2. individually isolated U937 colony forming cells was restored by partially Purified Molecules. Human milk LF and human serum TF were purified U937 growth activity. Partially purified U937 growth activity purchased from Calbiochem (San Diego, CA), affinity purified (11) and did not stimulate, enhance, or inhibit colony formation by normal human iron saturated (3, 11) as described elsewhere. Natural HuIFN-7 was bone marrow granulocyte-macrophage progenitors. U937 cells can thus purified to a specific activity of 2 x IO7NIH reference units/mg protein serve as a useful model for the study of growth regulation at the level of (17). a single cell. Preparation of PP-U937-GA. U937 cells (2 x 105/ml) conditioned McCoy's 5A medium (10% FBS) without or with 10~*M indomethacin for 24 to 96 h. Indomethacin was used to stop production of prosta- INTRODUCTION glandin E (15), which inhibits colony formation of U937 cells (14). CM Established hematopoietic cell lines have proven helpful in to be used for purification was prepared in the absence of indomethacin and concentrated 10 times using a Minicon-B-15 concentrator (Ami- evaluating factors that can influence the proliferation and dif ferentiation of normal and leukemia hematopoietic cells (1-4). con, Lexington, MA). One-mi fractions were loaded onto a G-100 superfine Sephadex column (Pharmacia Fine Chemicals, Piscataway, We sought to determine if an individual hematopoietic cell NJ) equilibrated with phosphate buffered saline, pH 7.0, and the eluted physically separated from other cells could proliferate and fractions were collected. The active material appeared to have a molec whether this proliferation could be suppressed and then restored ular weight greater than 100,000 and was purified > 1,000-fold by this by factors released from these cells. U937 cells, an established procedure (data not shown). human monoblast or early monocyte cell line (2, 5) were chosen Preparation of Cells for Colony Assay. Cells were suspended at 2 x for these studies. LF3 (3, 4, 6-10) and TF (11) have been 10s cells/ml in 5 ml of McCoy's 5A medium with 10% PBS and 10~* M indomethacin in Corning 25-cm2 T-flasks with 20 units HuIFN-7/ implicated as regulatory molecules for myelopoiesis and their ml. Cells were incubated at 37Â°Cin 5% CO2 in air under humidified actions in vitro on the suppression of the production/release of GM-CSF have been associated with the presence of MHC class conditions for 72 h, a time which allows for optimal induction of MHC II antigens on their respective target cells, monocytes-macro- class II antigens on U937 cells, U937 CFC, and responsiveness of U937 CFC to suppression by LF and TF (13). Received 12/20/85; revised 3/18/86; accepted 3/21/86. Colony Formation Assay. U937 cells were cultured in 1 ml of 0.3% The costs of publication of this article were defrayed in part by the payment agar (Difco Laboratories, Detroit, MI) culture medium (13). PP-U937- of page charges. This article must therefore be hereby marked advertisement in GA (10% v/v) was added with control medium, LF, or TF to the cells accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1These studies were supported by USPHS Grants CA 36740 (formerly CA which were plated at 500 cells/ml prior to gelling of the agar and 23528), CA 36464 (to H. E. B.) and CA 38661 (to B. Y. R.) from the National colonies were scored after 7 days of incubation. Aggregates containing Cancer Institute and by a Clinical Scholar Award from Sloan Kettering Institute more than 10 cells were scored as colonies, but colonies usually con to E.' To B. whom requests for reprints should be addressed, at Department of tained more than 30 cells. Colonies containing more than 100 cells were not unusual. Colony forming assays by IO5 nonadherent low Medicine, Indiana University School of Medicine, Clinical Building, Room 379, 541 Clinical Drive, Indianapolis, IN 46223. density normal human bone marrow cells were done using 5637 CM 3The abbreviations used are: LF, lactoferrin; CFU-GM, colony forming unit, (18) as described elsewhere (3,18). Colonies (>50 cells/aggregate) and granulocyte-macrophage; TF, transferrin; CFC, colony forming cells; HuIFN--y, clusters (3-50 cells/aggregate) were scored after 7 days of incubation. human -, interferon; CM, conditioned medium; CSF, colony stimulating factors; FBS, fetal bovine serum; MHC, major histocompatibility complex; PP-U937- Colony Assay for Single Isolated U937 Cells. Culture wells (24-well GA, partially purified U937 growth activity; GM-CSF, granulocyte-macrophage multiwell; Becton Dickinson, Oxnard, CA) contained 0.5 ml of media colony stimulating factors. composed of 40% double strength supplemented McCoy's media, 10% 3309

FBS, and 50% 0.6% agar. To the wells were added 10 Â¿<1ofcontrol These results strongly suggest that the restoring influence of media, 10 n\ of 5 x 10"* M LF (final concentration, 10~8M), without PP-U937-GA on colony formation suppressed by LF is not due or with 10 /il of PP-U937-GA. Individual cells were isolated by using a simply to inactivation of LF. hand controlled micropipet apparatus under an inverted microscope in Release of Growth Activity from U937 Cells. U937 cells were a sterile hood from the cells which had been suspended in media after preincubation for 72 h with HuIFN--y and washed 3 times. The presence allowed to condition culture medium for 1, 4, 24, 48, 72, and 96 h at concentrations of IO2, 2.5 X IO2, 5 x IO2, IO3, 5 x IO3, of a single cell was then verified in each well; if the cell could not be IO4, and 10s cells/ml in the presence of 10~6 M indomethacin. identified or if more than one cell was transferred, the individual well was discounted in the evaluation. In each experiment a 24-well control As shown in Table 3, medium conditioned by up to IO5 cells/ plate, LF plate, LF plus PP-U937-GA plate, and a plate containing ml for 1 and 4 h did not restore colony formation suppressed PP-U937-GA alone was set up. Colonies were counted after 7 days. by LF. Medium conditioned by IO5 but not IO4cells/ml for 24 Statistical Analysis. Three to 5 plates were scored for each sample, h, by IO3but not 5 x IO2cells/ml for 48 h, and by 5 x IO2but and the probability of significant differences between samples was not 2.5 x IO2cells/ml for 72 and 96 h, restored colony forma determined with Student's t test. Statistical analysis between the 10 tion of U937 cells suppressed by LF. For simplicity, even separate single cell experiments (Table 5) was performed using the though CM was prepared from IO2to 10s cells/ml, the data are Wilcoxon signed rank test. The mean Â±SE percentage of change from shown only for CM prepared from the lowest cell concentration control was derived from the values determined in the 10 separate experiments. Analysis of PP-U937-GA addition compared to control at each time point which restored colony formation and the media was made by the Wilcoxon-Mann-Whitney test and the Fisher next lowest concentration tested.. These experiments were re peated with CM from U937 cells preincubated for 72 h in exact test. suspension culture with IO"6 M indomethacin plus 20 units HuIFN-7/ml. The results, not shown, were equal to those given RESULTS in Table 3. Influence of PP-U937-G A on Cloning Efficiency of U937 Cells. In order to directly determine if LF was suppressing the U937 cells were preincubated for 72 h in suspension culture production/release of growth activity from U937 cells, U937 with 10~6M indomethacin plus 20 units HuIFN--y/ml, washed, cells preincubated for 72 h in suspension culture with 10~6 M and plated in semisolid agar culture medium at 500 cells/ml indomethacin and 20 units HuIFN-y/ml were washed and then with varying dilutions of PP-U937-GA and/or IO"8 M LF. One allowed to condition culture medium at 10s cells/ml in the of 3 reproducible experiments is shown in Table 1. PP-U937- absence or presence of 10~8 M LF. The CM was collected and GA has no effect by itself on the cloning efficiency of U937 preincubated at room temperature for 1.5 h with a 1:50 final cells, but dilutions of PP-U937-GA, as low as 10~6, restore dilution of the purified immunoglobulin fraction of rabbit anti- colony formation of LF-suppressed U937 cells. As shown in human LF in order to inactivate the carryover effects of LF on Table 2, for one of 2 reproducible experiments, PP-U937-GA U937 colony formation. This concentration of anti-LF inacti vates the effects of at least 10~7 M LF on suppression of the had no effect on colony formation by U937 cells in the absence of LF, but 10% v/v of 10-' and 10-' dilutions of PP-U937-GA release of GM-CSF from human mononuclear cells (3). The counterbalanced the suppressive effects of 10~13and IO"7 M LF. rabbit anti-human LF does not recognize TF immunologically Not shown is that the significant 50% inhibition of colony and does not influence the effects of concentrations as high as formation (P < 0.005) by pulse exposure of U937 cells to 10~8 10~7 M TF on suppression of the mitogen-activated release of M LF was completely counterbalanced by culturing the cells GM-CSF from human T-lymphocytes (11) (data not shown). with a 10% (v/v) IO'3 dilution of PP-U937-GA (P > 0.05). The U937 CM preincubated with anti-LF was then assessed for its capacity to restore colony formation of U937 cells Table l Titration ofPP-U937-GA on colony formation ofU937 cells suppressed by 10~8 M TF. As shown in Table 4, for one of 3 Colonyformation/500 reproducible experiments, 24-, 48-, 72-, and 96-h CM prepared Cells plated in presenceof cells in the absence of LF contained growth activity and restored 241 Â±7Â° Control medium colony formation suppressed by TF. Medium conditioned for PP-U937-GA (IO'1 dilution) 236 Â±8 -2 PP-U937-GA (IO-3dilution) 250 Â±6 +4 24 and 48 h in the presence of LF contained no detectable PP-U937-GA (IO"6dilution) 245 Â±9 +2 growth activity and did not restore colony formation suppressed PP-U937-GA (IO"7dilution) 247 Â±11 +2 LF(10-Â«M) 157Â±7 -35Â» by TF. Medium conditioned for 72 h in the presence of LF had LF (10-' M)+ PP-U937-GA(IO'1 dilution) 243 Â±12 +1 LF (10-' M)+ PP-U937-GA(KT3dilution) 243 Â±4 +1 Table 3 Influence of culture time and cell concentration on release from U937 LF (10'' M)+ PP-U937-GA(10-*dilution) 253 Â±14 +5 LF (10-' M)+ PP-U937-GA(10'' dilution) -39* cells of activities necessary to restore colony formation of U937 cells suppressed 147 Â±3 byLF â€¢MeanÂ±SE. *Significantchange from control mediumat P < 0.005. PreparationCMTime of U937 plated inagarwithout Table 2 Influenceof varyingconcentrationsofLF and PP-U937-GAoncloning MLFWithoutWithWithWithWithWithWithWithWithWithWithWithColonyformaorwith10~' tion/500 efficiencyofV937 cells (h)No tion/mlIO5IO5IO5Kr1IO35x cells133 Â±5"66 Colonyformation/500 CM(control)No Cells plated in presenceof cells %A CM142424484872729696Cellconcentra Â±467 Â±367 Control medium 129Â±3Â° Â±5130 PP-U937-GA (10-' dilution) 126Â±1 -2 Â±369 PP-U937-GA (IO'5 dilution) 126Â±4 -2 LF (IO'7 M) Â±1128 66 Â±6 -49Â» Â±568 LF (IO-13M) 72 Â±2 -44Â» IO25x Â±6128 LF (IO"7M)+ PP-U937-GA(IO'1 dilution) 122+ 3 -5 IO22.5 +268 LF (IO-7M)+ PP-U937-GA(10'' dilution) -1 xIO25x LF (IO"13M)+ PP-U937-GA (IO'1 dilution) 128Â±3 Â±1127 123Â±2 -5 IO22.5 Â±565 LF (IO-13M)+ PP-U937-GA (IO"5dilution) 129Â±6 0 x IO2CM Â±5%A-50Â»-50Â»-50Â»-2-48*-4-49*-4-49Â»-5-51Â» " Mean Â±SE. â€¢MeanÂ±SE. Â»Significantchange from control mediumat P < 0.005. *Significantchange from control (no Cm or LF) at P < 0.005. 3310

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. REGULATION OF SINGLE U937 CELLS Table 4 Influence ofLF on release from U937 cells of activities necessary to as marked as for colonies >30 or >40 cells. In each instance, restore colony formation ofU937 cells suppressed by TF however, the simultaneous addition of PP-U937-GA and LF U937 CM was prepared in the absence or presence of LF and preincubated with 1:50 final dilutions of purified immunoglobulin fractions of rabbit anti- restored cloning efficiency to control values. Addition of PP- human LF at room temperature for 1.5 h prior to adding to semisolid agar U937-GA to single cells in a well in the absence of LF had no cultures with or without 10"' M TF. significant effect on the cloning efficiency of U937 cells in 3 Preparation of U937CMWithout plated in agar Colony separate experiments. The cloning efficiencies for the 3 exper or with without or with formation/ LFNoTime (h) IO"1 M IO""%AWithoutWithWithWithWithWithWithWithWithWith168 M TF 500 cells iments in the presence of control medium were 52, 35, and 33% and in the presence of PP-U937-GA the values were, Â±13Â°100 CM(control)No respectively, 52, 48, and 35%. The second experiment with a 37% increase in cloning efficiency with PP-U937-GA did not CM2424484872729696WithoutWithWithoutWithWithoutWithWithoutWithCMÂ±7181 Â±988 have a significant increase and had P > 0.2 (two sided) by the Â±9171 Wilcoxon-Mann-Whitney test and P = 0.55 by the Fisher exact Â±393 Â±6172 test. Â±18123 Assay of PP-U937-GA for Activity on CFU-GM. PP-U937- Â±8165 Â±7168 GA was assessed at 10% v/v for ability to stimulate colony Â±4-40*+8-48*+2-45*+2-27*-20formation from CFU-GM in 10s nonadherent low density " Mean Â±SE. normal human bone marrow cells and to inhibit or enhance * Significant change from control (no CM or TF) at P < 0.005. colony formation stimulated by granulocyte-macrophage col ony stimulating factors present in 5637 CM. Without 5637 less growth activity than CM prepared in the absence of LF CM, no colonies or clusters formed in the absence or presence and only partially restored colony formation suppressed by TF. of PP-U937-GA. In the presence of 5637 CM, 41 Â±2 colonies LF did not suppress the release of growth activity into medium and 114 Â±5colonies and clusters formed. When PP-U937-GA conditioned by U937 cells after 96 h. These results indicated was added with 5637 CM, 41 Â±5colonies and 109 Â±5colonies that the suppressive effects of LF on production/release of and clusters formed; thus, PP-U937-GA had no stimulating, growth activity from U937 cells were not permanent. enhancing, or inhibiting effect on colony formation by CFU- Influence of LF and PP-U937-GA on Colony Formation at the GM. Level of a Single Cell. U937 cells preincubated for 72 h in suspension in the presence of IO""M indomethacin and 20 units DISCUSSION HuIFN-7/ml were washed and a single cell plated in 1 ml of medium/well in semisolid agar culture medium with McCoy's Isolated U937 cells can form colonies with the same cloning medium (control), 1(T8 M LF, 10 n\ of undiluted PP-U937-GA, efficiency as cultures containing 500 U937 cells/plate. Colony or 10-" M LF plus 10 /il of undiluted PP-U937-GA. A total of formation by U937 cells did not need exogenously added 10 separate experiments each was performed and the results of sources of growth factor. This fact plus suppression of colony colony growth in each category were added together and are formation by LF at the single cell level and restoration of LF- shown in Table 5. Eighty-seven of 190 Ã©valuablewellsof indi suppressed colony formation of single cells by growth activity vidual cells grown in the presence of control medium had present in PP-U937-GA suggest that U937 cells can serve as a colonies composed of >40 cells; this cloning efficiency of 46% useful model for studies of the control of growth at the level of corresponds to the cloning efficiency observed when cells are a single cell. cultured at 500/plate. In the LF wells, 57 of 197 wells grew In addition to evidence presented elsewhere (15), the wide colonies >40 cells for a cloning efficiency of 29%. The mean range of dilutions of PP-U937-GA (5 log units) that can over percentage of inhibition for 10 separate experiments was 40% come the suppression of colony formation by a wide range of Â±6% (SE); this is similar to the percentage of inhibition concentrations of LF (7 log units), offers strong evidence that obtained when the same concentration of LF is added to agar PP-U937-GA is not inactivating LF. Since LF does not block cultures containing 500 cells. When PP-U937-GA plus LF were the effect of the added growth activity yet suppresses colony added to the wells containing single cells, cloning efficiency formation, this argues against the possibility that colony for returned to control values; 83 of 175 Ã©valuablewellshad colo mation from a single cell plated in the absence of LF was due nies >40 cells for a cloning efficiency of 47%. For colonies to an earlier triggering event induced by growth factor(s) prior containing >30 cells, LF suppressed colony growth by 36% Â± to the cells being washed and plated in agar. If prior triggering 7%, simultaneous addition of PP-U937-GA and LF restored of proliferation was involved, single U937 cells should have cloning efficiency to control values (Table 5). Colonies >10 and formed colonies in the presence of LF with the same efficiency >20 cells were suppressed significantly by LF, but this was not as in the absence of LF.

Table 5 Influence of lactoferrin and PP-U937-GA on the cloning efficiency of single U937 cells (total of 10 separate experiments) ofControl Cells plated in presence M lactoferrin + lactoferrinNo. M mediumColony PP-U937-GANo.

size ofcoloniesNo. efficiency ofcoloniesNo. efficiency(%)2932 from con ofcoloniesNo. efficiency (no. ofcells)2:40 of cellsplated87/190 <%)46 of cellsplated57/197 trol Â±1SE-40 of cellsplated83/175 (%)47 Â±6" =Ã®30 90/190 47 64/197 -36 Â±7Â° 84/175 48 a20 100/190 S35710"* 75/197 38 -30 Â±6Â°-25 86/175 4955 >10No. 108/190Cloning 84/197Cloning 43%A Â±8*10'' 96/175Cloning Â°P& 0.005 (two sided) by Wilcoxon signed rank test. * P < 0.02 (two sided) by Wilcoxon signed rank test. 3311

The exogenous release of growth factor(s) is intimately de REFERENCES pendent on the number of U937 cells conditioning the culture 1. Koeffler, H. P., and Golde, D. W. Human myeloid leukemia cell lines: a medium and the time that the cells were allowed to condition review. Blood, 56: 344-350, 1980. the medium. Growth activity could be detected in medium 2. Ralph, P., Moore, M. A. S., and Nilsson, K. Lysozyme synthesis by estab lished human and murine histiocytic and lymphoma cell lines. J. Exp. Med., conditioned by a starting concentration of 500 cells/ml for 72 143: 1528-1533, 1976. h. No growth activity could be detected in media conditioned 3. Broxmeyer, H. E., Smithyman, A., Eger, R. R., Meyers, P. A., and deSousa, with a starting concentration of 250 cells/ml even after 96 h. M. Identification of lactoferrin as the granulocyte-derived inhibitor of colony stimulating activity (CSA)-production. J. Exp. Med., Â¡48:1052-1067, 1978. 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Interaction of lactoferrin, monocytes, and T-lymphocyte subsets in the regulation of steady-state granulopoiesis in vitro. J. Clin. growth activity from the subpopulations of U937 cells respon Invest., 68: 56-63, 1981. sive to its action. LF only decreases colony formation by ~50% 7. Yung, Y. P., and Moore, M. A. S. Long-term in vitro culture of murine mast over a wide range of LF concentrations and this was related to cells. III. Discrimination of mast cell growth factor and granulocyte CSF. J. Immunol., 729: 1256-1261, 1982. an action for a limited time on a cycling subpopulation of U937 8. Brown, R. D., Yuen, E., Rickard, K. A., Vincent, P. C., Young, G., and CFC with an increased density distribution of MHC class II Krogenberg, A. Plasma lactoferrin in patients with neutropenia. Blut, in press, 1986. antigens (13). Those colonies that form in the presence of LF 9. Peterson, V., Ambruso, D., Emmett, M., and Bartle, E. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. Regulation of the Proliferation of the Established Human Monoblast Cell Line, U937, at the Single Cell Level

Ellin Berman, Berish Y. Rubin and Hal E. Broxmeyer

Cancer Res 1986;46:3309-3312.

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