Regulation of HL-60 Differentiation by Lipoxygenase Pathway Metabolites I/I Vitro1

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[CANCER RESEARCH 50, 7257-7260. November 15, 1990] Regulation of HL-60 Differentiation by Lipoxygenase Pathway Metabolites i/i Vitro1

Alan M. Miller,2 Steven M. Kobb, and Raquel McTiernan

University of Florida, College of Medicine [A. M. M., S. M. K.J and the Veterans Affairs Medical Center [A. M. M., S. M. K., R. M.]. Gainesville, Florida 32610

ABSTRACT final ethanol concentration of 2%, a concentration found to have no effect on cell growth. Piriprost was dissolved in double-distilled H2O. We have studied the effects of lipoxygenase inhibition and metabolite Leukotrienes were provided by the Upjohn Co. and were prepared in addition on HL-60 cells induced to differentiate. When HL-60 are induced ethanol. by dimethyl sulfoxide (DMSO) in the presence of an inhibitor of lipox Leukemic Cells. HL-60 (12, 13) (American Type Culture Collection, ygenase, caffeic acid, there is a marked change from the expected Rockville, MD) was maintained in liquid culture in RPMI-1640 me phenotype of mature granulocytes to a population composed predomi dium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine nantly of mature monocytes. (DMSO alone: 54% granulocytes, 10% serum (Hyclone Laboratories, Logan, UT). For differentiation studies, monocytes; DMSO + caffeic acid: 23% granulocytes, 53% monocytes.) cells from log phase cultures were diluted to 2 x IO5 cells/ml and Addition of leukotriene D4to DMSO-induced, caffeic acid-inhibited cul cultured for 5 days. Inducing agents, lipoxygenase inhibitors, and tures resulted in a dose-dependent recovery of the granulocyte phenotype. leukotrienes were added at the time of plating. Addition of lipoxygenase inhibitors to phorbol ester-treated HL-60 cells Differentiation Induction. Cells were induced to granulocytic differ did not alter the expected monocytic differentiation. These results support entiation by the addition of DMSO (Sigma) to a final concentration of a role for leukotriene D4 in the regulation of granulocyte differentiation 1.3% (13). Monocytic differentiation was induced by addition of 10"' of HL-60 cells induced with DMSO. M TPA (Sigma) (14). Cell Characterization. Following incubation, IO5cells were placed on glass slides utilizing a Shandon cytocentrifuge (Shandon Southern, INTRODUCTION Sewickley, PA). Slides were stained with Wrights-Giemsa stain for An intact lipoxygenase pathway of metabolism is necessary morphology, Â«NAEfor monocytes, and ACE for granulocytes; all stains for granulocyte-monocyte progenitor cell proliferation and dif were obtained from Sigma. ferentiation (1-3). Growth of cells of myeloid leukemia origin Adherence. Cells (lO'/ml) were incubated in the presence of appro have also been shown to be dependent on an intact lipoxygenase priate inducers, inhibitors, and leukotrienes. Samples were set up in metabolic pathway (4-6). Growth of the human myeloid leu duplicate pairs. At the end of a 6-day culture period, one plate from kemia cell line, HL-60, is inhibited in a dose-dependent manner each pair was gently aspirated and the nonadherent cells were counted. The other member of each pair was vigorously scraped to remove by lipoxygenase inhibitors (4). Anthes et al. (7) have shown an adherent cells and the total cells were counted. The percentage of that an 8-fold increase in leukotriene synthesis occurs when adherent cells was calculated from the difference in cell count between HL-60 is induced to differentiate by DMSO,1 and Goerig et al. the two plates divided by the total count. (8) reported a rapid upregulation of 5-lipoxygenase metabolites when HL-60 is induced to differentiate. Ziboh et al. (9) have recently demonstrated that when HL-60 is induced to granu- RESULTS locytic differentiation there is a marked increase in LTD4 Effects of Lipoxygenase Inhibitors and Differentiation Induc synthesis, and when induced to monocytic differentiation there ers on HL-60 Growth. A concentration of caffeic acid known to is a marked decrease in the synthesis of LTC4 and LTD4, with have minimal effect on HL-60 growth was selected. Caffeic acid a significant increase in LTB4. To further examine the role of at 17.5 Ã•/Minhibited HL-60 growth after 5 days of culture by lipoxygenase metabolites in myeloid differentiation, we have approximately 25% (Table 1), DMSO alone inhibited by about examined the effects of lipoxygenase inhibitors and metabolites 40%, and TPA inhibited by 50%. The combination of caffeic on HL-60 cells induced to granulocytic or monocytic differen acid and DMSO caused additive inhibition (60%), whereas the tiation. combination of caffeic acid and TPA was no more inhibitory than TPA alone. MATERIALS AND METHODS Effects of Lipoxygenase Pathway Manipulation on HL-60 Differentiation. Addition of caffeic acid alone had no effect on Lipoxygenase Inhibitors and Metabolites. Inhibitors of lipoxygenase HL-60 differentiation as determined by morphological exami used in this study were piriprost (Upjohn Co., Kalamazoo, MI) (10) nation of Wrights-Giemsa-stained cytospins. As expected, and caffeic acid (Sigma Chemical Co., St. Louis, MO) (11). Caffeic DMSO treatment resulted in granulocytic differentiation, acid was prepared in ethanol and concentrated so as not to exceed a whereas TPA induced predominantly mature monocytes and Received l /8/90: accepted 8/9/90. macrophage differentiation. Caffeic acid alone did not appear The costs of publication of this article were defrayed in part by the payment to induce differentiation (Fig. 1). of page charges. This article must therefore be hereby marked advertisement in When caffeic acid was added to HL-60 cell cultures at the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by Public Health Service Grant CA44838 awarded by the National time of induction with DMSO, the cells that formed after 5 Cancer Institute, the Department of Health and Human Services, and the Medical days of incubation exhibited monocytic morphology on Research Service of the Department of Veterans Affairs. A. M. M is a recipient of an American Cancer Society Clinical Oncology Career Development Award. Wrights-Giemsa staining rather than the expected granulocytic Leukotrienes used in this study were provided by the Upjohn Co., Kalamazoo, morphology, with an increase in monocytes from 10 to 53% (P MI. < 0.001) (Fig. 1). When LTD4 (10~9 M) was added in addition 2To whom requests for reprints should be addressed, at Box 3-211. 3 Hillis Miller Health Center, University of Florida College of Medicine, Gainesville, FL to the DMSO and caffeic acid, there was less of a shift from 32610. 3The abbreviations used are: DMSO, dimethyl sulfoxide: LT, leukotriene; granulocytes to monocytes, with only 17% monocytes rather oNAE, a-napthyl acetate esterase; ACE. AS-D-chloroacetate esterase: PKC, pro than the 53% with DMSO and caffeic acid (P < 0.001) (Fig. tein kinase C: TPA. 12-O-tetradecanoylphorbol-13-acetate. 1). Similar results were obtained using piriprost as an alternate 7257

Table 1 Effect of lipoxygenase pathway manipulation on HL-6Ãœgrowth countat5 days0(x ConditionsNo 10')28 viable*93K6so7675 additionsCaffeicacid Â±3.0f21 (17.5iiM)DMSO Â±2.317 (1.3%)DMSO Â±3.111 acidDMSO+ caffeic Â±0.912 LTD4(5x+ caffeic acid + Â±1.1% IO'9 M)Cell " Cells are plated at a starting concentration of 1 x IO5cells/ml. All additions arc made directly to plates at the time of plating. h Viability determined as percentage of cells excluding trypan blue. ' Means Â±1 SD of 4 independent experiments.

O Blast -*- Promyelc Myelo -Â»-Meta o 1 1.0 g Bands + PMN LTD CONCENTRATION(r*Q â€¢Mono -t- Macro Fig. 2. Effects of leukotriene D4 on the morphology of HL-60 cells treated with DMSO and caffeic acid. All cultures contained DMSO (1.3%) and caffeic acid (17.5 /IM). LTD4 was added to cultures at time 0. After 5 days of culture, differential cell counts were performed on Wrights-Giemsa-stained cytospins. Points, means of 3 independent experiments: bars, Â±1SD.

Table 3 Histochemical and functional characterization of differentiated HL-60 Cells were plated at 1 x 10s cells/ml at the end of 5 days. Differential analysis of Wrights-stained cytospins. assessment of adherence to tissue culture flasks, and assays for iÂ«NAEand ACE were performed on all samples. Results are from 3-8 independent experiments and expressed as means Â±1SD. DMSO/ DMSO/CA/ DMSO +C-*. DMSO PIRIPROST CAÂ°-* LTD/ +C.A. +LTD. + DMSO Control DMSO TPA Fig. 1. Effects of caffeic acid (C.A.). piriprost. and C.A. + LTD., on DMSO- r; monocytes 1.3 Â±1.1 10.3 Â±4.8 53.1 Â±0.3* 17.6 Â±8.8" 58.6+18.8 induced differentiation of HL-60 cells. DMSO (1.3%). caffeic acid (17.5 ^M). % Â«NAE 2.0+1.0 7.0 Â±3.0 37.0 Â±15.0'' 12.0 Â±4.0'' 57.0+15.0 piriprost (20 JJ.M),and LTDÂ«(10~*M). were added to HL-60 cultures on day 0. % adherent 8.2 + 3.4 8.9 Â±7.2 33.0 Â±17.7' 20.9 Â±12.2' 24.9 Â±6.2 After 5 days of culture, differential cell count of Wrights-Giemsa-stained cytospins c', PMN 1.0 Â±0.4 53.8 Â±10.2 23.2 Â±10.2" 38.6 Â±16.7" 3.3 Â±2.6 was performed. Columns, means from 5-8 independent experiments: bars, 1 SD. % ACE 48 Â±15 71.8 Â±13 20.6 Â±6.0* 46.0 Â±6.0" 22.0+13.0 Promyelo. promyelocytes; Myelo, myelocytes; Meta, melamyelocytes; PMN, " CA. caffeic acid; PMN, polymorphonuclear neutrophil. polymorphonuclear neutrophils; Mono, monocytes; Macro, macrophages. * Results in DMSO/CA group are compared to DMSO-alonc group utilizing a two-tailed Student's t test. Paired analysis was utili/ed in analyzing adherence Table 2 Effect of lipoxygenase metabolites on HL-60 differentiation data, because those experiments were set up in parallel. ' Results in DMSO/CA/LTD4 group are compared to DMSO/CA group, as Cells plated at 1 x 105/ml and incubated for 5 days Â±additions. Differential above. analysis performed on Wrights-stained cytospin preparations; 5200 cells scored/ JP< 0.001. sample. % bands and ''Â¡monocytes and mature granulocytes macrophages Histochemical Analysis of HL-60 Differentiation. Confirma NoadditionDMSO 2.8Â°63.022.256.724.350.869.215.47.210.518.311.110.3SD.1.8 Â±0.31.5Â± (13%)DMSO 1.352.3 tion of cell phenotype was studied by the use of histochemical ^M)DMSO+ caffeic acid (17.5 Â±24.4I1.0Â± stains that preferentially stain monocytes (Â«NAE)or granulo LTA4DMSO+ caffeic acid + 1<0.02)(Table3). granulocyte differentiation, addition of lipoxygenase inhibitors resulted in marked cytotoxicity with only 25% viable by 5 days DISCUSSION and bizarre morphology of the remaining cells. We examined the ability of LTD., to affect the monocytic Changes in their arachidonic acid metabolic products, partic differentiation induced by TPA. No effect was seen when LTD., ularly the leukotrienes, are among the myriad of metabolic and was added to TPA-induced HL-60 cells (data not shown). molecular changes that occur as HL-60, a human myeloid 7258

leukemic cell line, differentiates from proliferating blasts and lipoxygenase activity was not studied; however, inhibition of promyelocytes to cells that express phenotypic and functional cyclooxygenase by indomethacin had no effect on differentia characteristic of mature granulocytes and monocytes (7-9). tion. In contrast, inhibition of lipoxygenase does effect differ Goerig et al. (8) reported increases in lipoxygenase metabolites entiation. Addition of LTD4 alone in the absence of DMSO or within minutes of differentiation induction. Ziboh et al. (9) in TPA-induced populations is not sufficient to cause granulo demonstrated marked differences in the leukotriene profiles of cytic differentiation, and therefore, additional events must be uninduced HL-60 cells and those induced to granulocytic dif triggered by DMSO that are not initiated by TPA. Other ferentiation by DMSO or monocytic differentiation by TPA. enzyme systems in addition to lipoxygenase must be stimulated. These differences may reflect another characteristic of the ma Included among these would be the previously reported activa ture cell function or may perhaps be essential intermediates in tion of a phospholipid and calcium-dependent protein kinase the differentiation process. In the current studies, we have (23) and activation of an Na+/H+ exchanger (24). The cell death attempted to clarify the role of the observed changes in lipox seen with retinoic acid in combination with lipoxygenase inhib ygenase metabolites by manipulating the pathway during the itors may represent toxicity of the combination. Alternatively, induction period. Inhibition of lipoxygenase metabolism during it may indicate that a different sequence of events is involved TPA-induced monocyte differentiation did not affect the ability when HL-60 is induced to granulocyte differentiation by reti of HL-60 cells to differentiate to monocytes. These results noic acid that does not include the necessary signals for mon suggest that lipoxygenase metabolites are not essential for ocytic differentiation. Distinct differences have been reported monocyte differentiation and that the reported increases in in the pathways to granulocytic differentiation induced by LTB4 levels are consistent with the known ability of mature DMSO and retinoic acid (25). Of interest is a recent report (26) monocytes to produce this metabolite (15). that acivicin, a glutamine antagonist, induces monocytic differ In contrast, when these cells are induced with DMSO in the entiation of HL-60. Among the many actions of acivicin is presence of a lipoxygenase inhibitor, there is a change from the inhibition of â€¢y-glutamyltransferase, the enzyme necessary for expected granulocytic phenotype to a predominance of cells the synthesis of LTD4 from LTC4. that are monocytic by morphological, histochemical, and func If the availability of LTD4 in addition to protein kinase C tional criteria. This shift does not represent a survival advantage activation were all that is necessary for granulocytic differentia of monocytes over granulocytes because there is a net 300% tion, one would expect that the addition of LTD4 to TPA- increase in the absolute number of monocytes in the DMSO induced cultures would be sufficient to induce granulocytic plus caffeic acid group as compared to the DMSO alone. These differentiation. We were unable to alter the TPA-induced mon data suggest that an intact lipoxygenase pathway is essential ocytic differentiation of HL-60 by addition of LTD4. One for the differentiation of HL-60 cells to granulocytes when possibility for this inability to affect differentiation is that induced by DMSO. Peptidoleukotrienes, particularly LTD.,, DMSO induces other events not involving the lipoxygenase were increased in the previous reports on lipoxygenase metab pathway that are essential for granulocytic differentiation. Al olism in DMSO-induced HL-60 cells (6, 8). In the current ternatively, it is possible that the lack of altered differentiation studies we have shown that the addition of LTD4 or its precur reflects the inability of LTD4 to get into cells and that entry sors to the cells exposed to both DMSO and a lipoxygenase into cells may be facilitated by DMSO. inhibitor resulted in a population that was predominantly gran In summary, an active lipoxygenase pathway and LTD4 syn ulocytic. In contrast, LTB4 had no affect under the same con thesis are necessary but not sufficient for the DMSO-induced ditions. granulocytic differentiation of HL-60 cells. Differentiation of These studies suggest that differentiation of HL-60 involves these cells to granulocytic and monocytic phenotypes share activation of multiple pathways. If the events that occur in some common pathways, and these may be combined with differentiation include lipoxygenation of arachidonic acid to other unique pathways that lead to the final differentiated LTDj, the cells express a granulocytic phenotype. If the same process. Recent studies demonstrate that the final phenotypic events are triggered with the exception of the lipoxygenase determination may occur late in the differentiation pathway pathway, they become monocytes. A common pathway that is (27). The current findings have been obtained with a single cell activated in both DMSO- and TPA-induced HL-60 cells is the line and inducing agent. However, they may provide insight activation of PKC (16, 17). DMSO may induce PKC activity into the processes that occur in determining the differentiated indirectly by stimulating the hydrolysis of polyphosphoinositide expression of a normal progenitor with multiple potentials, (phosphatidylinositol 4,5-bisphosphate), which releases diacyl- such as the granulocyte-monocyte colony-forming unit. glycerol, which can activate PKC (17, 18). TPA is reported to either work directly on PKC, substituting for diacylglycerol, or REFERENCES alternatively through phospholipase C (18, 19). It appears that DMSO must stimulate additional events that involve the syn 1. Miller. A. M., Ziboh. V. A., and Yunis, A. A. Evidence for involvement of thesis of LTDj and perhaps other lipoxygenase metabolites. the lipoxygenase pathway in CSF-induced human and murine myeloid colony LTD4 has been shown to have several effects on DMSO-induced formation. In: T. J. Towels, R. S. Bookman, K. V. Honn, and P. 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7260

Alan M. Miller, Steven M. Kobb and Raquel McTiernan

Cancer Res 1990;50:7257-7260.

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