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Differentiation of U-937 Histiocytic Lymphoma Cells Towards Mature Neutrophilic Granulocytes by Dibutyryl Cyclic Adenosine-3 ',5 '-Monophosphate1

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Differentiation of U-937 Histiocytic Lymphoma Cells Towards Mature Neutrophilic Granulocytes by Dibutyryl Cyclic Adenosine-3 ',5 '-Monophosphate1 (CANCER RESEARCH 50. 20-25, January I, I9TO] Differentiation of U-937 Histiocytic Lymphoma Cells towards Mature Neutrophilic Granulocytes by Dibutyryl Cyclic Adenosine-3 ',5 '-monophosphate1 Debra 1 . Laskin,2 Andrew J. Beavis, Andrea A. Sirak, Sean M. ()'( 'onncl 1,and Jeffrey D. Laskin Departments of Pharmacology and Toxicology. Rutgers University [D. L. L., A. J. B..A. A. S.J, Environmental and Community Medicine, UMDNJ-Robert Wood Johnson Medical School [J. I). L.], and (.'enter for Advanced Biotechnology and Medicine [D. L. L., S. M. O.J, Piscataway, New Jersey 08854 ABSTRACT of mature phagocytic cells and are not spécifietothe monocytic or granulocytic lineage. Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl Treatment of U-937 cells with agents that elevate intracellu- cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to lar cAMP such as dBcAMP, prostaglandin E2, or cholera toxin differentiate towards granulocytes. dBcAMP produced a dose- and time- has been reported to potentiate the differentiating effects of dependent inhibition of U-937 cell growth reaching a maximum after 48- both retinoic acid and vitamin D, (4, 13, 14). This suggests that h treatment with 500 /JM. At this concentration, dBcAMP had no effect cAMP-dependent phosphorylation reactions may modulate dif on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a ferentiation of myeloid cells (4). If cAMP is involved in differ concomitant increase in cells in the (.„<(.,phase.dBcAMP also induced entiation, then one would predict that agents that increase the appearance of f-met-leu-phe receptors on U-937 cells as well as the intracellular levels of this mediator would, by themselves, in ability to produce hydrogen peroxide and Superoxide anión.These data duce differentiation. In this regard, Kay et al. (16) reported that suggest that dBcAMP-treated U-937 cells were functionally mature. 1 mM dBcAMP induces the appearance of fMLP receptors and Using specific monoclonal antibodies and flow cytometry, we found that chemotaxis in U-937 cells. In contrast to this report, Olsson differentiated U-937 cells expressed the monocytic/granulocytic surface and his colleagues (13, 14) found that inducers of cAMP, markers MY8 and MAC-1, but not the monocyte specific markers MO2 including dBcAMP, were ineffective as differentiating agents or MY4. In addition, dBcAMP-treated U-937 cells did not stain for for U-937 cells. This is surprising since dBcAMP is known to nonspecific esterase, displayed less HLA-DR antibody binding than be a potent inducer of maturation in HL-60 cells (17). The undiffcrentiated cells and appeared smaller and more granular. These are all characteristics of mature granulocytes. Taken together our studies present studies were designed to evaluate the effects of phar indicate that differentiation of U-937 cells is not necessarily limited to macological concentrations of dBcAMP on maturation of U- the monocytic pathway of development. 937 cells. We found that dBcAMP in the range of 250-500 ¿IM rapidly induced differentiation of U-937 cells as evidenced by altered morphology, receptor expression and functional respon INTRODUCTION siveness. However, treated cells did not display nonspecific esterase activity or react with the monocyte specific markers, The U-937 cell line was derived from the pleural fluid of a MO2 or MY4. These data, together with our findings of de patient with diffuse histiocytic lymphoma (1). These cells have creased HLA-DR expression following dBcAMP treatment, been reported to differentiate into mature monocytes or mac suggest that U-937 cells are not necessarily committed to rophages when treated with TPA3 (2, 3), vitamin D., or its differentiate along the monocyte pathway and may have the metabolites (4-7), lymphokines including ->-Interferon (6, 8- capacity to mature into granulocytes. 11), or retinoic acid (12, 13). While all of these agents inhibit U-937 cell proliferation, the phenotypic response of the cells is MATERIALS AND METHODS variable and appears to depend on the inducer. For example, TPA-treated U-937 cells express markers characteristic of ac Chemicals. Propidium iodide, ferricytochrome C, dBcAMP, sodium tivated monocytes including HLA-DR and OKM, (2), are cy- butyrate, NBT, and fMLP were obtained from Sigma Chemical Co., totoxic and produce reactive oxygen intermediates (3). Retinoic St. Louis, MO. DCFH-DA and fNLLP-FITC were from Molecular acid-treated cells exhibit these functions as well as phagocytosis Probes, Eugene, OR, and TPA from LC Services Corp., Woburn, MA. (13, 14). Lymphokine treatment of U-937 cells also enhances The monoclonal antibodies, FITC-MY8 (IgG2.), PE-MY4 (IgG,), FITC-MO2 (IgM), and the isotypic controls. PE-IgG2>, PE-IgG,, and nonspecific esterase activity, induces the appearance of fMLP FITC-IgM were purchased from Coulter Immunology, Hialeah, FL. receptors and stimulates chemotactic responsiveness (9, 10, 11, MAC-1 antibody (IgG2b)was obtained from Hybritech Inc., San Diego, 15). While some of these responses such as nonspecific esterase CA. HLA-DR antibody (IgG2a)was from Becton-Dickinson, Mountain staining and expression of MO-2 or HLA-DR are unique to View, CA. FITC-conjugated goat-F(ab)'2-anti-rat IgG was from Jackson monocyte/macrophages, the morphological and functional re Immunoresearch, West Grove, PA. FITC-conjugated rat-F(ab)'2-anti- sponses observed in differentiated U-937 cells are characteristic mouse IgG was purchased from Southern Biotechnology Associates Inc., Birmingham, AL. Received .1/2/89; revised 8/1/89; accepted 10/2/89. Cell Culture. U-937 cells were kindly provided by Dr. Georgio The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Trinchieri at the Wistar Institute, Philadelphia, PA. Cells were main accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tained in suspension culture in RPMI 1640 medium supplemented with ' This work was supported by N1H grants AI20I83 and G M34310 awarded to 15% heat-inactivated fetal bovine serum, 2 mM L-glutamine (final D. L. L. 2To whom requests for reprints should be addressed. concentration, 4 mM), 100 units/ml penicillin, and 100 ¿<g/mlstrepto 3The abbreviations used are: TPA. ^-O-tetradecanoylphorbol-LVacetate; mycin. For our differentiation experiments, U-937 cells (1.25 x IO5) dBcAMP. dibutyryl cyclic adenosine-3',5'-monophosphate; cAMP, cyclic AMP; were resuspended in fresh culture medium in the presence of increasing fMLP. .V-formyl-melhionyl-leucyl-phenylalanine; NBT. nilroblue tetrazolium; concentrations of dBcAMP. The cells were then inoculated into tissue fNLLP, jV-formyl-.V-leucyl-leucyl-phenylalanyl-tyrosyl-lysine; FITC. fluorcscein culture flasks and incubated in a humidified atmosphere at 37°Cwith isothiocyanate: PK, phycoerythrin; DCFH-DA. 2'.7'-dichlorofluorescin diacc iate; SOD. superoxide dismutasc: RNase A. bovine pancreatic ribonuclease A; 5% CO2 in air for 24-96 h. HBSS, Hanks' balanced salt solution; PBS, phosphate buffered saline. Superoxide AniónRelease. Superoxide aniónrelease by U-937 cells 20 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. GRANULOCYTE DIFFERENTIATION OF U-937 CELLS was measured as the SOD-inhibitable reduction of ferricytochrome c ÃŒ|0K\-i18 (18). Cells (1 x IO6)were incubated at 37°Cwith 0.044 mvt ferricyto- IO 15 ' OX"~^ chrome c and increasing concentrations of TPA or control, in the '>ÄX ^-¿Sí3A^^^ presence or absence of SOD. After 30 min, the cells were centrifuged (300 x g, 5 min), the supernatants collected and absorbance at 550 nm 10-CONTROL ' determined spectrophotometrically. The amount of Superoxide anión CELLS/m dBcAMP (uM) formed is equal to the amount of ferricytochrome c reduced and was calculated (E= 21.1/nM/cm at 550 nm) using a baseline obtained from a sample containing SOD (34 ttg/m\). Duplicate samples were analyzed for each experiment. Results are presented as the mean ±SE of three experiments. Hydrogen Peroxide Production. Hydrogen peroxide production was 24 48 72 96 monitored by flow cytometry using DCFH-DA as previously described INCUBATION TIME (h) (19). Approximately 1 x 10* cells were incubated for 20 min at 37°C Fig. 1. Time-dependent inhibition of growth of U-937 cells by dBcAMP. U- with 5 UM DCFH-DA followed by incubation with TPA, fMLP, or 937cells(1.25 x lO'/ml) were treated with increasing concentrations of dBcAMP control for an additional 15 min. Green fluorescence intensity, which (•,50 UM;A, 125 JIM;A, 250 JIM;Ãœ,500 /JM; •¿1000JIM)or control (O) and allowed to grow for 96 h. At the indicated times, aliquots of cells were removed is directly proportional to hydrogen peroxide production, was then and enumerated using a Coulter Counter as described in "Materials and Meth analyzed by flow cytometry using a Coulter EPICS Profile equipped ods." Each point represents the mean ±SE from three separate experiments. with a 25-mW argon laser. For each analysis, 10,000-20,000 events Statistically significant (/' - 0.01) differences between treated and untreated I were accumulated. 937 cells are indicated by asterisks (ANOVA). The inset shows the effects of fMLP-Receptor Expression. U-937 cells (1 x IO6)were resuspended increasing concentrations of dBcAMP on growth of U-937 cells after 48 h in HBSS containing 1 /¿MfNLLP-FITC and incubated at 4°C.After treatment with the drug. 30 min, the cells were washed three times with HBSS and fixed overnight in HBSS containing 3% paraformaldehyde. The cells were 100i then washed, resuspended in HBSS and analyzed for green fluorescence by flow cytometry on the EPICS Profile. Innnunofluorescence. For indirect immunofluorescence analysis, U- 937 cells were washed three times in HBSS containing 0.1% gelatin or 10% serum, and then incubated with 200 ii\ of MAC-1 or HLA-DR antibody or the isotypic controls for 30 min at 4°C.Cells were then washed three times in HBSS and incubated with 200 ¿ilofthe appro priate FITC-labeled secondary antibody at 4°Cinthe dark.
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