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 spÃ©cifietothe 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 (.â€ž<(.,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 aniÃ³n.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 Â¿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 Â¿

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 37Â°Cwith 0.044 mvt ferricyto- IO 15 ' OX"~^ chrome c and increasing concentrations of TPA or control, in the '>Ã„X ^-Â¿SÃ3A^^^ 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 aniÃ³n 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 Â±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 37Â°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 (â€¢,50 UM;A, 125 JIM;A, 250 JIM;Ãœ,500 /JM; â€¢¿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 Â±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 /Â¿MfNLLP-FITC and incubated at 4Â°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 4Â°C.Cells were then washed three times in HBSS and incubated with 200 Â¿ilofthe appro priate FITC-labeled secondary antibody at 4Â°Cinthe dark. Thirty min CJ later the cells were washed three times and fixed overnight in 3% paraformaldehyde. For direct immunofluorescence experiments, cells were washed in gelatin or serum and then incubated with 200 Â¡AofPE- 0 24 48 72 96 MY4, FITC-MO2, FITC-MY8 or the appropriate isotypic control at 4'C. After 30 min the cells were washed and fixed as described above. INCUBATION TIME (h) All antibodies were used at 10 tug protein/ml. Cell-associated fluores Fig. 2. Effects of dBcAMP on cell cycle kinetics of U-937 cells. Cells were treated with 500 /iM dBcAMP and then fixed in 70"t ethanol. stained with cence was analyzed on the EPICS Profile flow cytometer. propidium iodide, and analyzed by flow cytometry as described in "Materials and Measurement of Cell Cycle. Cell cycle analysis was performed by Methods." The percentage of cells in each phase of the cell cycle was calculuted quantifying the DNA content of U-937 cells using the fluorescent, using the DNAFIT algorithm. Cytologie Software (Coulter Epics, FL). A, S intercalating agent, propidium iodide (20). U-937 cells (1.5 x 106)were phase; A, <â€¢,,<>, suspended in 4.5 ml of 70% ethanol in PBS. After 60 min at 4Â°C,the cells were washed and resuspended in 1 ml of propidium iodide staining 45% of undifferentiated U-937 cells were in the S phase of the solution (3.8 HIMsodium citrate, 0.01 mg/ml propidium iodide, and 0.5 mg/ml RNase A) and incubated at 4Â°C.Three h later the cells were cell cycle (Fig. 2). Treatment of the cells with 500 AIMdBcAMP produced a time-dependent decrease in the number of cells in washed, resuspended in PBS and analyzed on a Coulter EPICS 753 Dye Laser Flow Cytometer equipped with a 5-W argon laser adjusted the S phase reaching approximately 8% of control by 48 h. We to an output of 1000 mW at 488 nm. For each sample, 20,000 events also noted an accumulation of cells in the GU/G| phase of the were collected. cell cycle following dBcAMP treatment during this same time Nonspecific Esterase. a-Naphthyl acetate esterase (nonspecific ester period (Fig. 2). ase) activity was measured using a kit purchased from Sigma Chemical Inhibition of growth by dBcAMP was accompanied by alter Co. (St. Louis, MO). ations in U-937 cell morphology. After 48-h treatment with 500 uM dBcAMP, U-937 cells appeared smaller and more RESULTS irregularly shaped than undifferentiated cells (not shown). Treated cells also displayed numerous surface protrusions. Effects of dBcAMP on U-937 Cell Growth and Morphology. Staining with Wright-Giemsa revealed that the nuclear-to-cy- In our initial studies, we analyzed the effects of dBcAMP on toplasmic ratio was decreased in treated cells when compared proliferation of U-937 cells. We found that dBcAMP produced to control cells. In addition, the cytoplasm appeared more a dose- and time-dependent inhibition of growth of the cells granular. This change in granularity was correlated with an (Fig. 1). Growth inhibition was first evident 48 h after treatment increase in the 90Â°lightscatter property of the cells as measured of the cells with 250 Â¿Â¿MdBcAMPand reached a maximum by flow cytometry (not shown). Neither treated nor untreated after 96 h with 500-1000 MM. The effects of dBcAMP on U-937 cells adhered to the culture dishes. growth were not due to toxicity since cell viability, as deter Expression of Differentiation Markers on dBcAMP-treated mined by trypan blue dye exclusion, remained high (90-95%) U-937 Cells. A series of monoclonal antibodies, directed against at all time points and concentrations tested. To further char antigens expressed on maturing cells of monocytic or granulo- acterize the effects of dBcAMP on U-937 cell growth, we cytic origin, were used to characterize the effects of dBcAMP analyzed alterations in cell cycle using propidium iodide (20). on U-937 cells. By immunofluorescence and flow cytometry, During exponential growth we found that approximately 35- we found that cells treated with 500 MMdBcAMP for 48 h 21

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. GRANULOCYTE DIFFERENTIATION OF U-937 CELLS bound more of the antimonocytic/granulocytic antibodies MY8 and MAC-1 than did untreated cells (Table 1). However, neither treated nor untreated U-937 cells expressed the monocyte- Q- 80 specific markers recognized by the monoclonal antibodies MO2 or MY4. Interestingly, untreated U-937 cells were found to 60 O express high levels of HLA-DR antigen. Treatment of the cells Z o with dBcAMP decreased anti-HLA-DR antibody binding (Ta â€¢¿z.40 ble 1). m (fi To determine if U-937 cells express markers characteristic of 20 functionally active phagocytic cells, we next quantified binding of the fluorescently labeled chemotactic peptide fNLLP-FITC to the cells. We found that binding of fNLLP-FITC to U-937 O 24 48 72 96 cells increased with time following dBcAMP treatment (Fig. INCUBATION TIME (h) 3). Maximum expression of fMLP receptors was observed after 48-h incubation of the cells with 500 ^M dBcAMP. Untreated U-937 cells did not express fMLP receptors. Similar results >- have been reported by Pike et al. (21 ) and Kay et al. (16). We o also determined if differentiated U-937 cells displayed nonspe cific esterase activity. This enzyme has been used as a marker O for differentiated monocytes (9, 22). We found that neither 48- Lu h dBcAMP-treated nor untreated U-937 cells stained for non > specific esterase. The cells remained pale yellow when compared Iâ€” to monocytes and macrophages which stained dark brown or < LJ black (not shown). OL Production of Reactive Oxygen Intermediates by dBcAMP- treated U-937 Cells. A major functional response of mature 100 1000 monocytes and granulocytes is the production of reactive oxy LOG FLUORESCENCE INTENSITY gen intermediates (23). To determine if dBcAMP induced func Fig. 3. Effects of treatment of U-937 cells with dBcAMP on fMLP receptor tional maturation of U-937 cells, we quantified hydrogen per expression. U-937 cells were treated with 500 Â¿IMdBcAMP for 0-96 h. fMLP receptor expression was determined by immunofluorescence using fMLLP-FITC oxide and Superoxide aniÃ³n production by the cells. Using as described in "Materials and Methods." The percentage of cells binding fNLLP- DCFH-DA, we found that fMLP stimulated H2O2 production FITC was calculated using Overton's Cumulative Subtraction algorithm. Cyto by cells treated with dBcAMP. These effects were time-depend logie Software (Coulter Epics, FL). A. percentage of U-937 cells binding fNLLP- ent (Fig. 4) and dose-dependent (not shown) reaching a maxi FITC at various times after treatment with dBcAMP; B, single parameter histo gram showing the binding of fNLLP-FITC to control (unshaded curve) or 48-h mum in cells treated with dBcAMP for 72 h. TPA, a potent dBcAMP-treated U-937 cells (shaded curve). activator of phagocytes (23), was also found to stimulate H2O2 production by differentiated cells, although not as effectively as fMLP (data not shown). Pretreatment of the cells for 5 min with 10 //M catatase inhibited the production of H2O2 in re sponse to both fMLP (Fig. 5) and TPA (data not shown). In contrast to these results, undifferentiated U-937 cells did not z o produce H2O2 in response to either TPA or fMLP (data not o 6- shown). We also found that dBcAMP-treated U-937 cells produced significantly more Superoxide aniÃ³nthan did untreated cells as measured by reduction of ferricytochrome c (Table 2). Produc 2- tion of Superoxide aniÃ³n by differentiated U-937 cells in re sponse to TPA was dose-dependent reaching a maximum at 1.6 24 48 72 96 Table I Reactivity of U-937 cells with monoclonal antibodies to monocytic and INCUBATION TIME (h) granulocytic surface antigens Fig. 4. Effects of dBcAMP treatment on hydrogen peroxide production by U- U-937 cells, incubated with or without 500 n\i dBcAMP for 48 h, were 937 cells. Cells were treated with 500 JIMdBcAMP (â€¢)or control (O) for 0-96 analyzed for antibody binding by immunofluorescence and flow cytometry. Re h. Cells were then incubated with DCFH-DA for 20 min followed by 1 nM fMLP sults are expressed as mean fluorescence channel number. On our flow cytometer. or control. H2O; production was quantified 15 min later by flow cytometry. The each histogram has 256 channels which are displayed on a 3-decade log scale. data is presented as percentage stimulation of H2O2 production by fMLP over Each point represents the average of two samples Â±S.D. Control cells were control. treated with isotypic control antibodies. Cluster AntibodydesignationControlMY8ControlMAC-1U-937cell38.0 U-937cells40.01 MM(Table 2). Higher concentrations of TPA were cytotoxic. A Â±5.091.3 similar dose-dependent stimulation of Superoxide aniÃ³n pro Â±5.134.0 13.530.079.434.04.61.33.34.15.430.0duction was obtained with fMLP (data not shown). These Â±1.334.1 1ControlMO2 CD 1 Â±1.536.0 results were confirmed by light microscopy by monitoring the Â±7.129.0 ability of dBcAMP-treated cells to reduce NBT. Following TPA 14ControlMY4 CD +7.132.0 Â±5.531.0 stimulation, 89% of the dBcAMP-treated cells were found to Â±1.132.3 Â±0.932.6 14ControlHLA CD Â±1.632.0 Â±2.647.0 produce Superoxide aniÃ³n. In contrast only 7% of control cells Â±0.6101.0 Â±2.252.0 reduced NBT (data not shown). Pretreatment of the cells with DRUntreated Â±2.3Treated Â±3.3 SOD (0.8 mg/ml) inhibited TPA-stimulated reduction of NBT 22

ing decreased size and increased granularity, produce reactive oxygen intermediates and express several cell surface markers characteristic of mature myeloid cells. These include receptors for the bacterially derived chemotactic peptide, fMLP and the granulocyte/monocyte antigens, MY8 and MAC-1. However, these cells do not stain for nonspecific esterase, express the monocyte-specific markers MO2 or M Y4 and display decreased HLA-DR antigen expression. Taken together, these data sug gest differentiation of U-937 cells in response to dBcAMP is directed towards the granulocytic lineage. Inhibition of U-937 cell proliferation induced by dBcAMP 10 100 1000 was found to be time- and dose-dependent, reaching a maximum LOG FLUORESCENCE INTENSITY after 48 h treatment of U-937 cells with dBcAMP. This was Fig. 5. Inhibition of H2O2 production in dBcAMP-trcated U-937 cells by correlated with an accumulation of cells in the Gu/Gi phase of catatase. Cells were incubated with 10 UMcatalase or buffer and then with DCFH- DA for 20 min followed by l JIMfMLP or control. H2O2 production was quantified the cell cycle, and a decrease in the number of cells in the S 15 min later by flow cytometry and is directly proportional to log green fluores phase of the cell cycle. These changes in growth and cell cycle cence. .I. unsi iululated: I!. fMLP stimulated: C. unstimulated plus catalase: I). kinetics are characteristic of differentiating cells (24). Our find fMLP stimulated plus catalase. ings that the alterations in growth induced by dBcAMP were complete within 48 h are unique, since other inducers of U-937 Table 2 Effects of dBcAMP treatment on Superoxide aniÃ³nproduction by U-937 cells cell differentiation require 3-5 days to exert optimal effects (2, U-937 cells, incubated with or without 500 Â»iMdBcAMP for 48 h, were 5, 7, 13). A similar rapid inhibition of growth in response to incubated with ferricytochrome c and TPA or control in the presence and absence of SOD as described in "Materials and Methods."1 Superoxide aniÃ³nproduction dBcAMP has been reported previously in HL-60 myeloid leu was quantified spectrophotometrically 15 min later. Each point represents the kemia cells (17, 25). HL-60 cells are also known to differentiate mean Â±SE of three samples from three separate experiments. into granulocytes following dBcAMP treatment (17, 25). Superoxide aniÃ³nproduction Morphological and flow cytometric analysis revealed that cells)TPAconcentration(HM)00.161.6016.00160.001600.00UntreatedU-937cells0000.4(nmol/106 dBeAMP-treated U-937 cells were smaller than untreated cells and displayed increased granularity and numerous cytoplasmic U-937cells00.6 projections. These changes are distinct from those observed in U-937 cells treated with vitamin D3, lymphokines, retinoic acid, Â±0.10.2 or TPA (2, 4, 7, 10, 11, 13) and are consistent with maturing +0.13.2 granulocytic cells (12, 26). We also found that differentiation Â±2.4Â°8.3 Â±0.10.9 associated with dBcAMP treatment did not result in cell adher Â±0.61.4 Â±0.4Â°11.6 Â±0.6dBcAMP-treated + 0.6Â° ence or nonspecific esterase staining, two characteristics of Â°Statistically significant (P < 0.05) differences between untreated and mature mononuclear phagocytes (4, 9). U-937 cells have pre dBeAMP-treated cells (ANOVA). viously been reported to be weakly positive for nonspecific esterase and to exhibit increased esterase staining following in both differentiated and undifferentiated cells to 11 and 1%, induction of monocyte differentiation (9, 11). The differences respectively. in esterase staining observed may be due to the use of distinct Effects of Sodium Butyrate on U-937 Cells. We next analyzed subclones of U-937 cells (27). In this regard U-937 subclones the effects of sodium butyrate on growth and differentiation of have been reported to exhibit significant heterogeneity in their U-937 cells. As observed with dBcAMP, sodium butyrate (50- sensitivity to differentiating agents (4). 500 Â¿Â¿M)inhibitedproliferation of U-937 cells in a dose- and Previous studies have reported that untreated U-937 cells do time-dependent manner (data not shown). Growth inhibition not express antigens recognized by MAC-1 or HLA-DR anti was first evident 48 h after treatment of the cells with 500 pM bodies (11, 28, 29). In contrast, in our studies using flow butyrate and reached a maximum (35% inhibition) at 96 h. cytometry, we noted significant MAC-1 and HLA-DR antibody Sodium butyrate had no effect on viability at this time. Incu binding to untreated U-937 cells. Differentiation of the cells bation of the cells for periods longer than 96 h or with concen with dBcAMP was associated with enhanced binding of MAC- trations above 500 /Â¿Mrapidly induced cytotoxicity. Sodium 1, but decreased binding of anti-HLA-DR antibody. These butyrate treatment of U-937 cells also resulted in a 15% de findings are consistent with differentiation of U-937 cells to crease in the percentage of cells in S phase of cell cycle and a wards granulocytes. HLA-DR antigen is known to be expressed concomitant increase (20%) in cells in G0/G, phase (data not on most hemopoietic progenitor cells (30). During development shown). In contrast to dBcAMP, butyrate did not induce mor certain cell lineages retain this antigen (i.e., monocytcs and li phological or functional alterations in U-937 cells characteristic cells), while others (i.e., granulocytes), do not (31). HLA-DR of differentiated phagocytes. Cells treated with 500 ^M butyrate antigen expression on immature cells is also conserved in most for 48-96 h morphologically resembled untreated U-937 cells leukemias (32, 33). Our findings of high HLA-DR expression and did not express fMLP receptors or produce hydrogen on native U-937 cells provides further evidence for the imma peroxide (data not shown). ture nature of these tumor cells. As noted above, the variations in antigen expression observed in our studies may be due to differences in subclones of U-937 cells. In this regard, Gitter et DISCUSSION al. (27) have characterized a subclone of U-937 cells that is Our results demonstrate that U-937 histiocytic lymphoma HLA-DR positive, but esterase, peroxidase, and lysozyme neg cells can be induced to differentiate morphologically and func ative. These investigators suggest that this U-937 subclone may tionally by dBcAMP. U-937 cells treated with dBcAMP cease be arrested at an earlier stage of development. It is possible that to proliferate, display distinct morphological alterations includ the U-937 cell clone used in our experiments is similar to the 23

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. GRANULOCYTE DIFFERENTIATION OF U-937 CELLS clone described by Gitter and his colleagues. REFERENCES dBcAMP-treated U-937 cells also displayed evidence of func 1. Sundstrom. C., and Nilsson, K. Establishment and characterization of human tional maturation. These cells developed fMLP receptors and histiocytic lymphoma cell line U-937. Int. J. Cancer, 17: 565-577. 1976. could be stimulated to produce hydrogen peroxide and super- 2. Kolset, S. O.. Ivhed. I., Overvatn, A., and Nilsson, K. Differentiation- associated changes in the expression of chondroitin sulfate proteoglycan in oxide aniÃ³nin response to both fMLP and TPA. Interestingly, induced U937 cells. Cancer Res.. Â¥Â«.-6103-6108.1988. functional maturation of U-937 cells followed maximum 3. Balsinde. J.. and Mollinedo, F. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. Differentiation of U-937 Histiocytic Lymphoma Cells towards Mature Neutrophilic Granulocytes by Dibutyryl Cyclic Adenosine-3′,5′-monophosphate

Debra L. Laskin, Andrew J. Beavis, Andrea A. Sirak, et al.

Cancer Res 1990;50:20-25.

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