Document KJ61N6385eOkJREO95BOJ5yZ6

Benzene and Its Metabolite, Hydroquinone, Induce Granulocytic Differentiation in Myeloblasts by Interacting with Cellular Signaling Pathways Activated by Granulocyte Colony-Stimulating Factor Betsy A. Hazel, Annette 0'Connor, Rodica Niculescu, George E Kalf Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania,USA Key Words. Benzene Hydroquinone Granulocytic differentiation G-CSF LTDl Signal transduction Abstract. Chronic exposure of humans to benzene (BZ) causes acute myelogenous leukemia. These studies determined whether BZ, or its reactive metabolite, hydroquinone (HQ), affect differentiation of myeloblasts. BZ or HQ administered to C57BU6.l mice specifically induced terminal granulacyticdifferentiationof myeloblasts. The ability of the compounds to induce differentiation of the myeloblast was tested directly using the murine interleukin3 (IL-J)-dependentmyeloblasticcell line, 32D.3 ( G )and the human HL-60 promyelocytic leukemic cell line. Treatment of HL-60myeloblasts with BZ activated protein kinase C and upregulated the 5-lipoxygenase(LPO) pathway for the production ofleukotrieneD4(LTD,), an essential effector of granulocytic differentiation. Differentiation was prevented by sphinganine, a kinase C inhibitor, as well as by LPO inhibitorsand LTD, receptor antagonists. BZ and HQ also induced differentiation in 32D.3 (G)myeloblasts. Both compounds interact with cellular signaling pathways activated by granulocyte colony-stimulatingfactor (G-CSF) and thus replace the requirement for G-CSF. IL-3 induces a growth response, whereas G-CSF provides both growth and differentiation signals. BZ does not induce growthin the absence of IL-3, but provides a differentiation signal. Both HQ and LTD4induce differentiation and synergize with IL-3 for growth, however, neither support growth in the absence of IL-3. BZ-induced 32D cells showed a gradual progression of progenitor differentiation to granulocytes similar to that seen with G-CSF or LTD,. HQ Correspondence: Dr.George F. Kalf, Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Bluemle Life SciencesBuilding, Room 309,233 South Tenth Street, Philadelphia,PA 19107, USA. Received November 28, 1994; provisionally accepted February 16, 1995; accepted for publication February 28, 1995. BAlphaMed Press 10665099/95/$5.0010 STEM CELLS 1995;13:295-310 blocks differentiation at the myelocyte stage; only a small percentage of progenitors proceed to granulocytes. BZ, like G-CSF, upregulatesLTD4production, whereas HQ obviates the requirementfor LTD4 by activating the LTD4receptor. Introduction Benzene (BZ), a widely used industrial chemical and ubiquitous environmental pollutant, is a Class I carcinogen that causes acute myelogenous leukemia in humans that are chronically exposed [ 1-41. BZ hematotoxicity occurs when its hepatic metabolites [5,6], phenol, catechol and hydroquinone (HQ), are trans- ported to the bone marrow [7,81 and further oxidized in a peroxidase-mediated [9-111 reaction to biologically reactive intermediates that can potentially affect hematopoiesis. Because of the association between BZ exposure and an increased incidence of acute myelogenous leukemia, it is important to determine whether BZ and/or a metabolite, such as HQ, directly affect the stem cell and/or progenitor cells of the myeloid lineage. The ability to alter cytokine-dependent growth and differentiation in hematopoietic progenitor cells appears to be a property of agents with leukemogenic potential for humans [ 121. There have been several reports on the effects of BZ on hematopoietic stem and progenitor cells [131. In one study [141, a dose-dependent depression of all stem cell compartments was observed in BDFl mice exposed for 16 weeks to airborne concentrations of BZ as high as 99 ppm for 6 h per day, 5 days per week. However, the granulocyte-macrophage colony-forming unit (GM-CFU) was much less 296 Induction of Granulopoiesis by Benzene and Hydroquinone sensitive than the erythroid CFUs at higher doses for the kind gift of the 32D.3 (G) clone. The of BZ. Dempster and C. A. Snyder [151reported cells were maintained in Iscove's modified that short-term exposure of mice to BZ induced a Dulbecco's medium (IMDM) in the presence shift toward granulocytic differentiation, and a of 10% FBS and 3 u/ml recombinant murine growth advantage for granulocytic progenitor IL-3 (rMuIL-3) and supplemented with peni- cells in the marrow and spleen which increased cillin (50 IU/ml) and streptomycin (50 mg/ml). the total number of granulocytes. These results Cells were cultured at 37C in a 5%C 0 2atmos- suggest that BZ and/or HQ is acting on the phere with biweekly replacement of medium myeloid stem or progenitor cells. We report here and adjustment of cell concentration to lo5 that the administration of BZ or HQ specifically cells/ml for optimal growth. stimulates granulopoiesis in mice and induces All cell cultures were demonstrated to be granulocytic differentiation in myeloblasts of the free of mycoplasma contamination by periodi- human promyelocytic leukemic cell line, HL-60, cally testing the culture supernatant with a and the normal murine interleukin 3 (IL-3)- radiolabeled mycoplasma cDNA probe. dependent myeloblastic cell Line, 32D.3 (G). They do this by replacing the requirement for G-CSF and leukotriene D4(LTD,), respectively, for induction of differentiation. Reagents IMDM,RPMI 1640, Dulbecco's phosphate buffered saline (PBS) minus Ca2+and Mg2+, Tyrodes buffered salt solution (TBBS) and PenlStrep were obtained from Mediatech, Materials and Methods Washington, D.C. BZ (spectroanalyzed) and HQ were purchased from Fisher Scientific Co., Animals C57BU6J inbred male mice were obtained from Jackson Laboratories, Bar Harbor, ME at 6 weeks of age (18-20 g). Animals were housed at an AAALAC-approved central animal facility (73" f 3F; humidity 53%; 12 h light cycle) in polycarbonate cages (four mice per cage) with hardwood bedding and fed Purina Rodent Chow and water ad libitum. Animals were acclimated for 1 week and weighed between 22 and 25 g when used. The experiments reported were carried out under a protocol approved by the Institutional Animal Care and Use Committee. Pittsburgh, PA. FBS, the AS-D Chloroacetate Esterase Assay Kit, LTD,, sphinganine, caffeic acid, pepstatin, leupeptin, phenylmethylsulonyl fluoride (PMSF), rhodamine-labeled antimouse immunoglobulin, nitroblue tetrazolium (NBT), May-Grunwald/Giemsa stain and acivicin (aamino-3-chlor-4,5-y~5-isoxazole acetic acid) were obtained from Sigma Chemical Co., St. Louis, MO. The 5-lipoxygenase inhibitor, AA861, was a product of Wac0 Bioproducts, Richmond, VA. The LTD4 receptor antagonists were gifts from Merck Frosst Canada, Inc., Quebec (MK571)and Eli Lily & Co., Indianapolis, IN (LY 163443). Monoclonal antibody against human L-12-2 granulocyte-specificsurface antigen was a kind gift from Dr. Giovani Rovera. Recombinant human G-CSF, (rHuG-CSF) and rMuIL-3 were obtained from R&D Systems, Minneapolis, MN. Giemsa blood stain, Azure Type B, was obtained from EM Diagnostics, Gibbstown, NJ and HemaQuick II from the Curtin Matheson Co., Houston, TX. Gen Probe, San fetal bovine serum (FBS).Cells used in the exper- Diego, CA was the source of the Mycoplasma 1 iments were from passages 18-42.The IL3depen- Test Kit. 32Piwas a product of Dupont NEN, dent myeloblastic cell line, 32D.3, was derived Boston, MA. from normal bone marrow of C3WHeJ mice by Greenberger[161.It has a normal karyotype and is Exposure of Cells to HQ, BZ, G-CSF, LTD, 1 nonleukemic [16]. The clone was further charac- and Other Agents terized for IL-3 dependence by Metcalf[171, and HL-60 cells (5 x 105/ml)were incubated for G-CSF induction of differentiation by Valtieri with or without BZ in RPMI 1640/10% FBS for et al. [181 who gave the clone the designation 7 days at 37C after which differentiationto gran- (G). We are indebted to Dr. Giovanni Rovera ulocytes was assessed. 32D cells (2.5 x 10S/ml) Hazello'Connor~iculesculKalf were incubated in IMDM/IO% FBS with or without BZ, and with or without 3 u/ml rMuIL-3. When HQ was used, the cells were pretreated with 2 pM HQ in PBS/2 mM glucose (PBS-A) for 30 min at 37C after which the cells were collected, washed twice with PBS and placed into culture in IMDM/lO% FBS with or without 3 u/ml rIL-3. G-CSF and LTD, were added directly to the culture medium. The lipoxygenase inhibitors and LTD, receptor antagonists were added directly to the culture medium in amounts indicated in the legends to the tables and figures 20 min before the addition of the inducing agent. After 3 days of incubation, a sample of each culture was removed for cell counting and the remainder of the culture was diluted to 2.5 x lo5cells/ml to insure optimal growing condi- tions. Unless indicated otherwise in the figure or table legend, incubation was continued for 4 days, after which the cells were analyzed for the granulocytic phenotype. Assessment of Differentiation Granulocytic differentiation was assessed by: 1) the acquisition of granulocytic morphology, specifically promyelocytes, metamyelocytes and segmented or mature cells, 2) the development of superoxide production as measured by the reduction of NBT, 3) the development of chloroacetate esterase activity, and 4) in the case of HL-60 cells, the appearance of L-12-2 granulocyte specific surface antigen. Morphological assessment of differentiation was performed by cytospin preparation and May Grunwald/Giemsa staining. Percentages were based on the average obtained by counting 200 cells on each of triplicate slides. For NBT reduction, cells were incubated for 1 hour with 0.125 nmol/l TPA and 0.05% NBT. Cells were fixed on a slide, counterstained with safranin and the percentage of cells containing black granules of reduced NBT was determined. Chloroacetate esterase activity was determined by incubating fixed cells with a diazonium salt, AS-D chloroacetate and Red Violet at pH 6.3 for 5 min at 3 7 T , after which the percentage of cells containing red granules was determined. The presence of L- 12-2 surface antigen was detected by fluorescence microscopy using rhodaminelabeled antimouse immunoglobulin against the monoclonal antibody. 297 Treatment of Animals with BZ and HQ Mice received BZ (600m a g body weight) in corn oil or HQ (25 or 50 mg/kg body weight) in PBS i.p. twice per day, 7 h apart, for 2 days. Controls received corn oil or PBS. Eighteen h after the final injection the animals were killed by cervical dislocation, their femurs were removed and the bone marrow cells were obtained as described under the determination of bone marrow cellularity and morphologic hematology. The concentrations of BZ and HQ were not cytotoxic and the viability of the cells flushed from the femurs was >98%as measured by Trypan Blue exclusion. Determination of Bone Marrow Cellularity and Morphologic Hematology The epiphysial plate on each end of the femur was removed and 1 ml of undiluted FBS was forced through the femur using a syringe with a 25-gauge needle. The marrow was collected in a tissue culture tube and a single cell suspension was prepared by passing the marrow through the syringe two additional times. A sample (100 p1) of the cell suspension was used to prepare a slide which was stained as described below and used for a differential cell count. The remainder of the cell suspension was diluted in cold lysing buffer (10 mM Tris-HCI, pH 7.4, 155 mmoM NH,Cl) to remove red blood cells. The number of nucleated cells was determined with a hemocytometer and viability was tested by Trypan Blue exclusion. Viability was > 98%. Staining was carried out as follows: the slide was dipped in Wright-Giemsa stain (Hema Quik 11) for 3 min, washed by dipping in tap water for 3 min and allowed to air-dry for at least 30 min. Counter staining was carried out by immersing the slide for 7 min in Giemsa (Azure B Type) prepared fresh as a 1:50 dilution in Hydrion buffer, pH 6.8. The slide was then washed for 30 sec with running tap water and allowed to airdry. Differential cell counting was carried out using oil immersion at 1 0 0 ~F.ive hundred cells per slide were counted and the percentage of each type of cell present was determined. BZ-Induced Protein Kinase C Phosphorylation of HL-60 Cellular Proteins HL-60 cells ( 3 x 10Vml) were twice washed in TBBS-Ca2+to remove P, from inside the cells and then preloaded with 32P(,1.5 mCi/ml; sp. act. 1 Ci/mmol) by incubation for 1 h at 22C 298 Induction of Granulopoiesisby Benzene and Hydroquinone in calcium and phosphate-free TBBS. The cells were washed three times with TBBS and incubated in TBBS-Ca2+with 5 mM BZ and/or 40 pM sphinganine or TBBS for 5 min. When sphinganine was used it was added 10 min before the other reagents. The reaction was stopped by chilling the tubes on ice, the cells were recovered and lysed in 100 pl of a solution containing Tris-EDTA, pH 8.0, 2 mM PMSF, 0.1 mM pepstatin A, 0.1 mM leupeptin and 0.01% Triton X-100. Protein was determined by the method of Bradford [191. Polyacrylamide Gel Electrophoresis Lysate protein (8 pg/well) was loaded onto an 0.8 mm, 8% T vertical slab polyacrylamide gel. Electrophoretic separation of proteins was canied out at 150V for 3.5 h. The gel was silverstained, dried and exposed to X-OMAT AR film for 96 h at -7O'C. HPLC Analysis of LTD4Formation in HL-60 Cells Cells (1.5 x 106/ml in PBS) were incubated with or without 5 mM BZ or with BZ and 1 pM AA861 for 24 h at 37C. After removal of the cells, a 100pl sample of the incubation medium was extracted with an equal volume of toluene and the organic phase evaporated to dryness.The residue was suspended in 100 pl of mobile phase (65:35:0.02 vlvlv methano1:water:acetic acid, pH 5.6) and 50 pl was injected into a reverse phase, 5 mm ODS HPLC column. An LTDl standard (1 pg) was run as a control. Statistical Analysis Data between groups were analyzed using the t-test or ANOVA followedby Dunnett's t-test. Results are expressed as mean values f SD. A p 5 0.01 was considered significant. Results Stimulation of Granulocytic Differentiation in Mice by BZ and HQ As can be seen in Figure 1, the administration of BZ to mice stimulated the differentiation of myeloblasts as measured by an increased percentage of promyelocytes and intermediate granulocytic progenitors in the bone marrow one day after the last BZ injection (three days after the initiation of treatment). BZ had no effect on the az BLAST PM GB OS Fig. 1. The induction of granulocytic differentiation in C57BL16J mice by BZ or HQ. Groups of mice (n = 4) were injected with BZ (600 mgkg body weight) or HQ (25 or 50 mgkg body weight) twice daily for 2 days. The control animals received corn oil and PBSA. Eighteen h after the final injection the mice were killed, the bone marrow obtained and a morphological analysis of the bone marrow carried out as described under Methods. Data are presented as absolute numbers of myeloid cells per femur represented by blast cells and total granulocytic cells of 500 cells analyzed. Qualitatively similar results were obtained when the data were expressed as the percent of nucleated bone marrow cells. The results are expressed as the mean i SD (n = 4); * p SO.001 as compared with the control group. The data from various cell types from the individual marrows are extremely tight (they range from SD f 0.001 to f 0.03) and too small to be plotted by the printer/plotter so no error bars are visible. PM, promyelocytes; GBand, myelocytes and metamyelocytes (ring) forms; Gseg, segmented (mature granulocytes). number of myeloblasts. The number of mature granulocytes (G seg) was not stimulated at Day 3, however, in experiments carried out for 7 days, BZ significantly increased the number of mature granulocytes compared to control values (data not shown).The experiment presented is representative of three such experiments which gave similar results. Identical results were obtained when the data were expressed as percentage of total cells counted. HQ administered to mice for 3 days at a dose of 50 m g k g body weight also stimulated granulocytic differentiation as indicated by an increased percentage of promyelo- cytes and metamyelocytes (Fig. 1). In contrast to BZ, HQ doubled the number of myeloblasts and stimulated differentiation to band forms to a greater extent than BZ, and the terminal differentiation of intermediate progenitors (band forms) to mature (segmented) granulocytes was i1 Hazel/O'Connor/Niculescu/Kalf 299 ,M E 0CONTROL BENZENE system with which to study the mechanism of induction of differentiation, we turned to the use of myeloid cell lines. The well-studied human HL-60 promyelocytic leukemic cell line [20], which consists of approximately 35 to 45% myeloblasts and the remainder promyelocytes, was chosen because it has been used in many studies as a surrogate for the GM-CFU,and a number of agents have been shown to induce ter- minal granulocytic differentiation in these cells [20]. The normal mouse IL-3-dependent 32D myeloblastic cell line was used because it has Fig. 2. BZ-induced granulocytic differentiationof HL60 myeloblasts. Cells (5 x 1Wlml)were cultured with and without 5 mM BZ as described under Methods for up to seven days. The developmentof the granulocytic phenotype was determined by measuring the characteristics presented in the figure. The values represent the mean i SD of at least three experiments where been adapted to terminally differentiate to granulocytes in response to G-CSF [18], and because data obtained with this cell line can be compared with data obtained in the mouse. As can be seen from the data presented in Figure 2 and Table I, exposure of HL-60 myeloblasts to BZ followed by incubation for 7 days resulted in terminal granu- each sample was carried out in triplicate. locytic differentiation measured by morphology *Significantly different from the control cells at p s 0.001. (determination of the percent of cells classified as promyelocytes or higher granulocytic forms) t and the development of several characteristics of the granulocyticphenotype. No significant induc- limited. HQ administered at 25 mg/kg body tion of nonspecific esterase activity indicative of weight caused a lesser, but significant stimulation monocytes was detected nor was monocyte mor- of granulopoiesis (data not shown). At these phology observed (Table I). The ability of BZ to doses of BZ or HQ,there was no loss of animals, induce granulocytic morphology as well as the no overt signs of toxicity and the viability of the appearance of other markers of the granulocytic cells flushed from the femurs remained at greater phenotype was a function of the concentration of than 98%. the BZ used over the range 0.1 mh4 to 5 mM (data not presented). As can be seen in Table I, the HL- Induction of Granulocytic Differentiation in 60cell population consists of about 45% promye- Myeloblasts by BZ and H Q locytes and 35% myeloblasts. BZ caused a The results of the in vivo experiments sug- significant decrease in the number of myeloblasts gested that BZ per se, or by metabolism to HQ, is as well as promyelocytes, and a corresponding capable of inducing granulocytic differentiation. shift into more intermediate progenitors and However, they do not indicate whether either mature granulocytes. The majority of the differ- compound causes induction directly or indirectly entiated progenitors appeared to be metamyelo- in vivo. To answer this question and to have a cytes and myelocytes, but a significant number Table I. Morphological assessment of benzene-induced granulocytic differentiation of HL-60 cells System Blast Cell Type (76) "Mature" Total Promyelocyte Myelocyte Metamyelocyte Granulocyte Granulocyte HL-60 36 45 2 15 HL-60 + 5 mM BZ 15 29 16 30 2 19 10 56 Cells were treated with benzene (BZ) as described in Figure 2. Morphological assessment was performed by Cytospin preparation and staining with May-GriinwaldlGiemsastain. Percentages were based on the average obtained by counting 200 cells on each of triplicate slides in six different experiments. 300 Induction of Granulopoiesis by Benzene and Hydroquinone of terminally differentiated granulocytes was observed (Table I). BZ-Induced Activation of Protein Kinase C and the Inhibition of Granulocytic Differentiation in HL-60 Cells by the Kinase C Inhibitor, Sphinganine Activation of protein kinase C is involved in the induction of differentiation in HL-60 cells by tetraphorbol acetate (TPA) and other agents [20]. BZ has been shown to activate protein kinase C in intact rabbit platelets as well as the partially purified enzyme from mouse brain [21]. Consequently we tested whether BZ-induced differentiation of HL-60 myeloblasts to granulocytes was dependent on protein kinase C activation and therefore should be inhibited by sphinganine, a potent and specific inhibitor of protein kinase C in cell systems and in vitro [22]. In the representative experiment presented in Figure 3, HL-60 cells were pre-exposed to 32Ptio label ATP and treated with 5 mM BZ under conditions that induce granulocytic differentiation. The activation of protein kinase C was measured by the phosphorylation of cellular proteins as monitored by SDS-PAGE analysis and autoradiography (Fig. 3). BZ induced the transfer of 32Pi from AT[32P]to a number of HL-60 proteins (lane a) that was prevented by preincubation of HL-60 cells with 40 pM sphinganine prior to BZ treatment (lane b). The phosphorylation observed with BZ (data not shown) was equally as great as that observed in other experiments which utilized HL-60 cells treated with 100 ng TPA as a positive control. Protein kinase C on plasma membranes from BZ-treated HL-60 cells was capable of lipiddependent phosphorylation of the known protein kinase C substrate, histone 111-S (data not shown). Sphinganine also prevented BZ-induced granulocytic differentiation (Fig. 4), but by itself did not affect differentiation (data not shown). Since both BZ-induced phosphorylation of cellular proteins and granulocytic differentiation were inhibited by the kinase C inhibitor, the results implicate BZ activation of protein kinase C in BZ-induced granulocytic differentiation in HL-60 myeloblasts. The Involvement of the 5-Lipoxygenase (LPO)Pathway in BZ-Induced Granulocytic Differentiation An active LPO pathway of metabolism that converts arachidonic acid to the peptidoleukotriene,LTD4,is essential for normal [23,24] as well as leukemic [25,26] myeloid progenitor 129 loo 89 52 44 38 27 20 17 ae +v) mN Fig. 3. Autoradiograph of an SDS-polyacrylamide gel separation of "P-labeled cellular proteins from BZ-treated HL-60 cells. Cells (3 x 106/ml)were preloaded with 3zP,for 1 h, washed and exposed to 5 mM BZ (lane a), BZ plus 40 pM sphinganine (lane b) or buffer (lane c ) for 5 min as described under Methods. Lysate protein (8 pg/lane) was added to each lane of an 0.8 mm, 8% T polyacryamide gel. Electrophoresis was carried out at 150 V for 3.5 h. The gel was silver stained, dried and exposed to x-ray film for 96 h at -70C. Hazel/O' ConnorlNiculesculKalf 301 0100 CONTROL BENZENE BENZENE 6 SPHINGANINE 1lLnl 0 MORPHOLOGY WT L122ANTlGEN REDUCTION 5.01 L 5.03 A UntreatedH L M ) Cell1 U 02-TrWt.d ~ BZ + LPO lnhibhor Fig. 4. Inhibition of BZ-induced granulocytic differ- LTC, LTD, entiation in HL-60 cells by sphinganine. Cells ( 5 X I IOVml) were preincubated with 40 pM sphinganine in RPMI 1640/10%FBS for 10 min at 37'C. 5 % C02. Fig. 5. BZ-induced formation of LTD, in HL-60 cells. The cells were exposed to 5 mM BZ and assessed for Cells (1.5 x 106/ml in PBS) were incubated with or granulocytic differentiation after incubation for seven without 5 mM BZ (HPLCprofiles 2 and 3) or with BZ days as described under Methods.The values repre- and 1 pM AA861 (profile 4) for 24 h at 37'C. After sent the mean i SD of at least three experimentswhere removal of the cells, a 100pl sample of the incubation / each sample was canid out in triplicate. *Significantly medium was extracted with an equal volume of toluene different from the control cells at p IO.OO1. and the organic phase evaporated to dryness. The ( residue was subjected to HPW analysis for the presence of LTD, as described under Methods. Retention times cell proliferation and differentiation; LTD, is an intermediate in CSF-induced clonal growth of (min) are printed above the peaks. An LTD, standard (1 pg) was added as a control (profile I). GM-CFU [23, 241. The ability of BZ to cause the production of LTD, and its immediate pre- cursor, L E 4 , under conditions where BZ induced myeloblasts by BZ and stopped its constitutive granulocytic differentiation in HL-60 cells was production in the promyelocytes (Fig. 5). AA- investigated. A lipid extract of the culture 861 and another inhibitor, caffeic acid, prevented medium of differentiating HL-60 cells grown in BZ-induced granulocytic differentiation (Table the presence and absence of a 5-LPO inhibitor II). The inhibition was reversed by the addition of was subjected to reverse-phase HPLC. In sev- LTD4concomitantly with the inhibitor. A highly eral experiments, LTD, was produced in BZ- specific LTD, receptor antagonist, MK-571, [28] treated cells in amounts two- to four-fold greater also completely inhibited BZ-induced granulo- than in untreated cells. Figure 5 presents an cytic differentiation in HL-60 myeloblasts, and HPLC profile from a typical experiment in which this inhibition was prevented by the concomi- BZ induced the formation of LTD, (two-fold tant addition of LTD, (Fig. 6). Similar results increase over untreated cells) in HL-60 cells. (data not shown) were obtained with another LTD4is present in the untreated HL-60 cell pop- receptor antagonist, LY 163443 [29]. ulation (Fig. 5) because approximately half of The conversion of the immediate precursor, the cells are promyelocytes that have aborted LTC,, to LTD, requires the removal of a glutamyl their differentiation at that stage and thus con- group from LTC, by y-glutamyl transpeptidase tain LTD,. The remainder are myeloblasts in [30], a reaction that is inhibited by the glutamine which BZ should induce LTD, formation. The antagonist acivicin [23]. Data presented in Table requirement for BZ-induced formation of LTD, El show that 1 pM acivicin prevented BZ-induced for granulocytic differentiation was demonstrated differentiation to granu1ocytes.This inhibition by showing that 5-LPO inhibitors prevented both could be overcome by the addition of LTDl to BZ-induced LTD, production and granulocytic the culture medium, further supporting a role for differentiation in HL-60 myeloblasts (Table 11). LTDI in BZ-induced differentiation of HL-60 The highly specific 5-LPO inhibitor, AA-861 cells to granulocytes. Taken together, these 1271, prevented the formation of LTD4induced in results suggest that BZ, via activation of protein 302 Induction of Granulopoiesis by Benzene and Hydroquinone Table 11. Effect of 5-lipoxygenase inhibitors on benzene-induced granulopoiesis System Morphology NTB Reduction Percentage of cells counted Control plus BZ plus Bncaffeic acid plus Bncaffeic acid/LTD4 plus BZ/AA86 1 plus BZ/AA861/LTD4 12.3 i 5.5 37.5 f 3.8' 17.5 i 3.9 37.1 i 3.7b 23.7 2 3.3 54.7 f 10Sb 16.7 i 3.5 45.7 f 3.3" 20.8 i 3.7 42.8 f 8.3b 18.3 i 1.7 28.7 i4.1b Cells (5 x IOVml) were preincubated in culture with 5-lipoxygenase inhibitors caffeic acid (100 pM),AA861 (1 pm) and with LTD, ( I pM) in RPMI 1640115%FBS for 10 min at 37'C, 5% C02.BZ (5 mM) was added directly to the culture and NBT reduction and granulocytic differentiation were determined after incubation for seven days. Values represent the mean f SD of at least two experiments carried out in triplicate. ND, not determined. 'Significantly different from control and BZ-treated cells pretreated with inhibitors. bSignificantly different from cells treated with BZ and inhibitors. p S 0.01 kinase C, induces the formation of LTD4required for the initiation of granulocytic differentiation. Induction of Granulocytic Diferentiation in the Myeloblastic Cell Line 320.3 (G)by BZand H Q In order to compare the induction of granulocytic differentiation by BZ and HQ with that of the normal physiological inducer, G-CSF, we turned to the IL-3-dependent, 0COMROL BENZENE BENZENE 6 MK-571 BENZENE 6 MK-571 ** NET REDUCTION MORPHOLOGY Fig. 6. Inhibition of BZ-induced granulocytic differentiation in HL-60 cells by a 5-LPO inhibitor. Cells (5 x 1OVml)were preincubated with 1 pM MK-571 in RPMI 1640/10%FBS or IO min at 37"C,5% CO,. BZ (5 mM) was added and granulocytic morphology was assessed after incubation for seven days. Values represent the mean i SD of two experiments where each sample was carried out in triplicate. *Significant difference from the control cells at the p 50.01 level; **significant difference from cells treated with BZ and MK-57 1, but no significant difference from cells treated with BZ only p S 0.01. diploid, myeloblastic cell line, 32D.3 (G) which was derived from normal mouse bone marrow and adapted to differentiate in the presence of G-CSF [ 181. Table IV presents data from a representative of four experiments which gave similar results.There are few differentiated myeloid cells in the presence of IL-3 alone, which, although obligatory for proliferation and survival of the myeloblast, does not induce differentiation. rHuG-CSF that is fully capable of binding to the mouse G-CSF receptor induced terminal differentiation to granulocytes. Substitution of noncytotoxic concentrations of BZ or HQ in place of G-CSF also induced differentiation in 32D myeloblasts (Table IV). While both BZ and HQ replaced the requirement for G-CSF for differentiation to granulocytes, neither BZ (Fig. 8A) nor HQ (Fig. 8B) was able to obviate the dependence of the cells on IL-3 for survival and growth. The Ability of LTD, to Induce Granulocytic Differentiation in 3 2 0 Myeloblasts Since LTD4 is a downstream effector for G-CSF-induced signal transduction, and BZ has been shown to produce LTDl via activation of protein kinase C and thus arachidonic acid release, we tested the ability of LTD, to replace G-CSF, BZ and HQ in the induction of granulocytic differentiation in 32D myeloblasts. As can be seen in Figure 7, LTD, is capable of replacing these inducing agents in a concentration-dependent induction of terminal differentiation in 32D myeloblasts. HazeYO'Connor/Niculescu/Kalf 303 Table IJI.Effect of the y-glutamyl transpeptidase inhibitor, acivicin on benzene-induced granulocytic differentiation System Morphology NTB Reduction Percentage of cells counted Control plus BZ plus BZIacivicin plus BZ/acivicin/LTD4 12.3 f 5.5 37.5 f 3.8' 22.0 f 1.0 45.3 f 11.9b 16.7 f 3.5 45.7 f 3.3' 23.5 f 4.2 47.3 f 9 . l b Cells (5 x 1O%nl) were preincubated in culture with acivicin (1 pM) and LTD4in RPMI 1640/15% FBS for 10 min at 37'C. 5% C02.BZ (5 mM) was added directly to the culture and NBT reduction and granulocytic differentiation were determined after incubation for seven days. Values represent the mean f SD of at least two experiments carried out in triplicate. "Significantly different from control and BZ-treated cells pretreated with acivicin. bSignificantlydifferent from cells treated with BZ and acivicin.p I O . 0 0 1 . 1 I I' i ii Effects of Inhibitors of LTD, Formation and of LTD, Binding to Its Receptor on the Ability of H Q to Induce Granulocytic Differentiation in 3 2 0 Myeloblasts The induction of differentiation by G-CSF is inhibited by a specific 5-LPO inhibitor (Table V.A.), as would be expected since G-CSF Table IV. Induction of granulocytic differentiation in the mouse myeloblastic cell line, 32D.3 (G)b,y benzene and hydroquinone System1 Morphology' NTB Reduction Percentage of cells counted IL-3 alone IL-3 + G-CSF IL-3 + BZ IL-3 + HQ 7.5 f 3.2 30.8 2 2.5 53.0 f 1.0 67.8 f 3.3 4.3 f 0.6 36.7 f 1.7 55.0 f 0.7 66.8 f 1.4 ICells (2.5 x I(Ylm1) were pretreated with HQ (2 pM in PBS-A) or PBS-A for 30 min at 37C.The cells were harvested by centrifugation, suspended in IMDM (2.5 x IO5cellslml) containing 3 u/ml rMuIL-3 and 10%FBS. rG-CSF (0.15 ng/ml) or BZ (5 mM) were added to the PBS-A-pretreated cells. Controls and HQ-pretreated cells received only PBS-A. After three days of culture at 37T, 5% C02,the medium was changed and the cells diluted to the original concentration to insure optimal growth conditions.The incu- bation was continued for four more days at which time parameters indicative of granulocytic differentiation were assessed. The values listed represent the mean f SD of the results in triplicate wells. Data is presented as the percentage of cells showing granulocytic differentiationout of a total of 200 cells counted. 'Includes cells from promyelocytic progenitors to terminally differentiated granulocytes. causes the release of arachidonic acid, the substrate for 5-LPO, from plasma membranes [31]. The ability of HQ to induce differentiation is not prevented by a 5-LPO inhibitor suggesting that HQ, in contrast to G-CSF and BZ, does not induce the formation of LTD4,but rather functions in some other way, perhaps by interacting with the LTD, receptor. An experiment was carried out to ascertain whether the ability of HQ to induce terminal granulocytic differentiation in 32D myeloblasts could be prevented by an LTD, receptor antagonist. The addition of the specific antagonist, MK571, completely blocked HQ- induced terminal granulocytic differentiation (Table V.B.). It is possible that HQ interacts with the ligand-binding domain of the receptor to activate its signal and initiate the cascade of events that result in granulocytic differentiation. Interaction of Differentiation-Inducing Agents with IL-3 for Growth of 3 2 0 Myeloblasts In the hematopoietic system, cytokines, unlike growth factors in other systems, induce cell growth that is coupled to differentiation such that clonal expansion of a differentiating progenitor cell population is observed. As can be seen in Figure 8A, BZ does not provide a growth signal for 32D myeloblasts in the absence of IL-3, nor does it synergize with IL-3 to promote growth. G-CSF also does not synergize with IL-3 to promote growth, but is capable of inducing growth in the absence of IL-3 (Fig. 8A). Neither LTD, nor HQ can induce a signal for proliferation in 32D myeloblasts in the absence of IL-3 (Fig. 8B). In the presence of IL-3, both LTD4 and HQ showed a significant stimulation of cell , i I f it 304 Induction of Granulopoiesisby Benzene and Hydroquinone OJ o iuo iod 1 0 7 iod 1 0 5 LTD, CONCENTRATION (M) 20 15 10 2 uwa 5 "9 0 12 34 Fig. 7. LTD,-induced granulocytic differentiation in 32D myeloblasts. Cells (2.5 x I(y/ml) in IMDM (3 u/ml rIL-3; IOBFBS) were incubated with or without LTD, for three days at 37'C. 5% COI. Cells were thinned to the original cell number/ml and incubated an additional 3 days without the further addition of LTD,. Cytospin slides were stained for morphological analysis using May-Griinwald/Giemsastain. Cells were monitored for superoxide production by following NBT reduction using TPA/NBT and counterstaining with Safranin. 12 3 DAYS POST TREATMENT 4 5 growth which, in the case of HQ, was more than 100%at day 4. A Comparison of the Kinetics of Stage-Specific Granulocytic DifferentiationInduced by the Physiological Inducers G-CSF and LTD,, and by BZ and H Q These agents all transmit a signal for differentiation in the presence or absence of IL-3. In the presence of the minimal concentration (3 u/ml) of IL-3 required for survival and proliferation of 32D myeloblasts, each of the inducers of granulocytic differentiation caused 100% total differentiation, but the degree of differentiation was delayed in comparison with cells grown in the absence of IL-3 (Table VI). The proliferative signal provided by IL-3 competes with the differentiation signal from the inducer such that the degree of both the total and terminal differentiation is decreased at 6 days. In the absence of IL-3, G-CSF, in contrast to BZ, HQ or LTD4, can provide signals for proliferation and differentiation. Consequently, G-CSF alone (Table VI, line 3) caused a level of terminal differentiation comparable to that observed with the other inducers in the absence of IL-3; however, the rate at which 100% total differentiation was achieved was Fig. 8. Effects of differentiation-inducingagents on proliferation of 32D cells in the presence or absence of IL-3. A) Cells (2.5 x 10Yml) were incubated in IMDM/IO% FBS with 5 mM BZ or rG-CSF (500 dml) with or without rIL-3 (3 u/ml) for eight days at 37'C, 5% C02.B) Cells were pretreated with 2 pM HQ/PBS-A or PBS-A for 30 min at 3732, 5% COz, washed twice with PBS-A. PBS-A-treated cells were also treated with LTD, (3.8 pM).AI1 cultures were incubated for eight days without further feeding. Cell viability and number were determined every second day. Data points represent the mean of triplicate samples which were within 5% of each other. Data were normalized to the controls. delayed (Table VI). A comparison of the data presented in Table VI (total differentiation versus terminal differentiation on day 6 in the absence of IL-3) is presented in Figure 9. G-CSF induced predominantly, terminally differentiated granulocytes whereas HQ induced predominantly myelocytes. BZ and LTD4produced a significant number of terminally differentiated granulocytes as well as an increased number of intermediate progenitors. IL-3 alone induced virtually no differentiation. An analysis of the kinetics of stage-specific granulocytic differentiation was performed on 32D myeloblasts induced by the physiological Hazel/O'Connor/Niculescu/Kalf 305 Table V. The effects of a 5-lipoxygenase inhibitor (AA861) and an LTD4receptor antagonist (MK571) on G-CSF- and HQ-induced differentiation of 32D cells System Morphology NTB Reduction Percentage of cells counted A. Control +G-CSF +G-CSF + 5-LPO inhibitor +HQ +HQ + 5-LPO inhibitor 17.5 f 2.6 70.8 f 3.5 31.9i0.9 81.5 f 1.3 66.1 i 1.7 12.5 f 3.6 48.2 f 10.6 12.6f 2.4 48.6 f 5.7 38.6 f 2.0 B. Control +G-CSF +G-CSF + R antagonist +HQ +HQ + R antagonist 17.5f 2.6 70.8 f 3.5 *32.8 i 8.8 81.5 1.3 24.1 i 1.8 12.5 f 3.6 48.2 f 10.6 26.8 f 6.2 48.6 f 5.7 15.2 f 2.5 Cells (2.5 x 105/ml) were pretreated with a final concentration of 2 pM HQ as described in the legend to Table IV with the addition of the 5-LPO inhibitor, AA861, or the LTD, receptor antagonist, MK571, at 1 pM final concentration. Morphological analysis was performed as described in the legend for Table IV. l and chemical inducers in the absence of IL-3.This allowed a study of the effects of inducer in the absence of any proliferative effects of IL-3.G-CSF caused a slight increase in each of the granulocytic progenitor stages at day 2 (Fig. 10A). Because of its ability to provide a proliferative signal as well as one for differentiation, G-CSF showed a marked stimulation of promyelocytes at day 4 as well as mature granulocytes. By day 6, the number of promyelocytes had decreased as was expected because of matu- ration and because the proliferative signal only permits two or three replicative cycles. The number of intermediate forms was comparable to the number at day 4 and the number of mature granulocytes was greater than 50%.By Table VI. Effects of inducing agents on patterns of granulocytic differentiation Agent IL-3 IL-3 + G-CSF G-CSF IL-3 + BZ BZ IL-3 + LTD4 LTD4 IL-3 +HQ HQ Day 0 0.5 i 0.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 Percent of Total Differentiation Day 2 0.0 f 0.0 2.0 f 0.0 19.3i 0.4 3.3 f 0.3 19.0i 2.2 9.2 f 3.2 32.0 i 0.0 4.3 f 1.4 40.0 f 0.5 Day 4 0.7 f 0.3 24.0 i 2.0 71.520.9 22.3 f 1.6 89.8 i 1.1 30.2 i 2.8 97.0 f 0.5 41.8 f 2.3 91.5 f 0.0 Day 6 6.0 * 0.0 91.8 f 1.6 90.8 f 1.6 86.8 f 2.3 99.7 i 0.6 92.8 f 1.3 99.3 i 0.3 88.5 f 2.8 98.8 2 0.3 Percent of Terminal Differentiation Day 6 1.o 16.7 51.2 18.0 39.8 25.2 46.8 14.8 17.2 Cells (2.5 x 105/rnl)were treated as described in the legend for Figure 8. Samples of each culture were collected every second day and cytospin preparations were made and stained. Morphologic analysis and determinationof the percentages for stage-specific differentiation were performed as described for Figure 9. Percentage of total differentiation was calculated by totaling the percentages of stage-specific differentiation for each sample. Terminal differentiation was calculated based on the number of segmentedhanded granulocytes over the total number of cells counted for each sample on day 6. I 306 Induction of Granulopoiesisby Benzene and Hydroquinone PM MY ME MA Fig. 9. Granulocytic differentiation in 32D cells 6 days post-treatment with inducing agents. Cells (2.5 x 105/ml)were treated with inducing agents as described in the legend for Figure 8. Samples of each culture were taken on day 6 and Cytospin slides were prepared in triplicate for each culture and stained for morphological analysis with May-Griinwald/Giemsa as described under Methods. Two hundred cells were counted and an average percent for each stage of differentiation was determined. PM, promyelocytes; MY, myelocytes; ME, metamyelocytes;MA, mature segmentedhanded granulocytes. day 8, almost all of the early progenitor forms had terminally differentiated to granulocytes. In comparison, LTD,, a downstream effector of G-CSF signaling, was incapable of providing a proliferative signal as evidenced by the lower number of promyelocytes detected over the sixday period (Fig. 10B). There were many more intermediate progenitors (myelocytes) and terminally differentiated granulocytes at day 2 in the presence of exogenous LTD,, most probably because the cells do not have to wait for LTD, to be produced following the G-CSF signal. At day 4, the promyelocyte burst seen with G-CSF is absent and the percentage of intermediate and mature forms (45%) is significantly increased because the effector was added directly and there was no proliferation signal. By day 6, the myelocytes have begun to mature into metamyelocytes and the number of terminally differentiated granulocytes has remained constant, probably because those that were mature at day 4 are beginning to die off. Because of the lack of a proliferation signal and the rapid induction of w t BZ-TREATED I I DAYP DAY^ DAY^ DAY^ 8 P f:70 HQ-TREATED D40 LLi10 0 PYMM. P DAYP DAY^ DAYG DAY^ Fig. 10. Profile of stage-specific differentiation induced by G-CSF (A), LTD4(B), BZ (C)and HQ (D). Cells (2.5 x 10Vml) were treated as described in the legend for Figure 8. Samplesof each culture were collected every second day and Cytospin preparations made and stained. Morphologic analysis and determination of the percentages for stagespecific differentiation were performed as described for Figure 9. Percentage of total differentiation was calculated by totaling the percentages of stage-specific differentiationfor each sample. P, promyelocyte; My, myelocyte; M, metamyelocyte; Ma, mature granulocyte. HazeUO'Connor/Niculescu/Kalf differentiation in the presence of LTD4, there were few viable cells by day 8; however, those that remained were 100% terminally differentiated (data not shown). Thus, while the pattern of differentiation with G-CSF or LTD4was different, they had in common the fact that at each day, the major progenitor type was the terminally differentiated granulocyte. BZ induced a pattern of differentiation at day 2 that was qualitatively similar to that seen with G-CSF or LTD, (Fig. IOC). At day 4, BZ showed a pattern of differentiation similar to LTD, except that the predominant progenitor type was the myelocyte rather than the terminally differentiated granulocyte. At day 6 the BZ-induced pattern continues to be similar to LTD, except that the numbers of myelocytes and metamyelocytes are higher. In contrast, HQ induced an incomplete differentiation pattern; at each day studied, HQinduced differentiation appears to be partially arrested at the myelocyte stage (Fig. 10D). Thus, BZ appears to slow the progression of granulocytic differentiation in 32D myeloblasts beyond the myelocyte stage at day 4 which resolves itself at day 6 (Fig. lOC), whereas HQ appears unable to induce significant differentiation beyond the myelocyte stage (Fig. 10D). Here, as in the case of LTD,-induced differentiation, the number of viable cells present at day 8 was very low, but those present were mature granulocytes in the case of BZ and with HQ, a mixture of predominantly intermediate progenitors and some mature granulocytes. Discussion Exposure of C57BU6J mice to a dose of BZ known to depress other hematopoietic cell lineages [32] significantly stimulated granulocytic differentiation as well as the total number of granulocytes indicating that granulopoiesis was also occurring (Fig. 1). BZ did not provide a growth signal for myeloblasts, but stimulated their differentiation to promyelocytes and intermediate progenitors. It did not stimulate the production of mature granulocytes at day 3, however, when measured at day 7, it had increased the number of segmented, mature granulocytes (data not shown). The results showing a general increase in granulocytes after BZ exposure are consistent with those showing an increase in the number of granulocytes in bone 307 marrow of DBN2J mice after short-term exposure to BZ [151. HQ, a major metabolite of BZ found in the bone marrow, also induced growth and differentiationof granulocyticprogenitor cells measured at day 3 when administered to C57BU6J mice (Fig. 1) as well as in mouse 32D myeloblasts in culture (Fig. 8B). In contrast to BZ, HQ provided both growth and differentiative signals for myeloblasts that increased the numbers of all pmgenitor forms, but appeared incapable of inducing terminal differentiation (Fig. 1) for reasons that are not clear at the present time. In order to study the mechanism(s)whereby BZ and HQ stimulate granulopoiesis, we investigated whether the inductive effect of BZ or HQ on granulopoietic differentiation could be reproduced in myeloblasts in culture. BZ, at noncytotoxic concentrations, caused a dose-dependent specific induction of terminal granulocytic differentiation in HL-60 myeloblasts (Table I, Fig. 2), as measured by the determination of granulocytic morphology and functional parameters of granulocytic differentiation such as superoxide production, chloroacetate esterase activity and the appearance of the specific surface antigen, L- 12-2. BZ induction produced a majority of intermediate progenitors (metamyelocytes and band forms), but a significant number of mature granulocytes were observed (Table I). This corresponds to what has been previously observed with HL-60 cells, that granulocytic differentiation induced by several agents is somewhat incomplete and defective [20]. BZ is known to activate protein kinase C [21]. Exposure of HL-60 cells containing 32Plabeled ATP to BZ resulted in the 32P-phosphorylation of cellular proteins (Fig. 2) under conditions where BZ induces granulocytic differentiation. The activation of kinase C appears to be involved in the induction of granulocytic differentiation of BZ-treated HL-60 myeloblasts, since the phosphorylation of cellular proteins and the induced differentiation were prevented by the concomitant presence of BZ with the specific kinase C inhibitor, sphinganine (Fig. 3). Differentiation of HL-60 myeloblasts to granulocytes by BZ appears to involve the induction of a functioning 5-LPO pathway for the production of LTD, since the differentiation is inhibited by the 5-LPO inhibitors, caffeic acid and AA-861 (Table II), by the specific LTD, receptor antagonist, MK-571, (Fig. 6) and by interruption of the LPO anabolic pathway at i" 1 ! i I 308 Induction of Granulopoiesis by Benzene and Hydroquinone the LTC4step by acivicin, an inhibitor of y-glutamyl transpeptidase (Table 111). Inhibition by all of these agents was prevented by the concomitant addition of LTD, with the agent. These results together support the view that the role of BZ in inducing granulocytic differentiation in HL-60 myeloblasts is to produce LTDl that is a necessary, if not a sufficient, signal for granulocytic differentiation. BZ appears to function in a manner similar to the physiological inducer of granulocytic differentiation, G-CSF. Although G-CSF has not yet been reported to cause the production of LTD4,it has been shown to cause the activation of phospholipase A2and the release of arachidonic acid from cell membranes [31]. BZ is also capable of inducing granulocytic differentiation in the diploid IL-3-dependent myeloblastic cell, 32D.3 (G)derived from nor- mal mouse bone marrow (Table IV). While IL-3 induces only a growth response, rHuG-CSF in the absence of IL-3 provides both growth (Fig. 6) and differentiation (Table V) signals for 32D myeloblasts. BZ is unable to induce growth in the absence of IL-3, but can provide a differentiation signal. Neither G-CSF nor BZ synergizes with IL-3 in stimulating growth. HQ also induces granulocytic differentiation in 32D myeloblasts (Table V). BZ and HQ can replace the requirement for G-CSF for induction of differentiation. It should be pointed out that in the experiment reported in Table V, no effort was made to optimize the concentration of a given inducer so that the magnitude of the BZ-or HQinduced differentiation cannot be quantitatively compared with that of the physiological inducer, G-CSF. The results, however, do indicate that BZ and HQ can induce granulopoiesis in 32D mouse myeloblasts since both the number of progenitor cells and terminally differentiated granulocytes increased, and they support our in vivo results on the induction of granulopoiesis by BZ and HQ in mice (Fig. 1). The ability of HQ to synergize with GM-CSF to stimulate GM-CFU has been studied by Irons et al. [33], who showed that pretreatment of nonadherent murine bone marrow or lineage-restricted hematopoietic cells with HQ in vitro, followed by culture in complete medium for 8 days, significantly enhanced the number of granulocytemacrophage colonies induced by recombinant GM-CSF. Optimal enhancement was observed with 1 pM HQ, was largely independent of the concentration of GM-CSF, and was not observed with other marrow metabolites such as phenol or catechol. The facts that BZ-induced granulocytic differentiation in myeloblasts in culture is prevented by inhibitors of protein kinase C and 5-lipoxygenase support the view that BZinduced differentiation is due to BZ per se and not the result of its metabolism to HQ in the myeloblast. Consequently, not all of the granulopoietic activity of BZ in vivo can be attributed to its metabolism to HQ. During chronic BZ exposure, BZ and HQ are present in the bone marrow and consequently both can contribute to granulocytic differentiation. On the basis of results present here, BZ, like G-CSF, appears to activate the arachidonic acid cascade and upregulate the 5-LPO pathway for the production of LTD4. HQ appears to obviate the requirement for LTD, by activating the LTDl receptor since specific LTD4 receptor antagonists, but not 5-LPO inhibitors, prevent HQ induction of granulocytic differentiation in myeloblasts. One can speculate about the roles of BZ and HQ in BZ-induced acute myeloid leukemia. As a known clastogen, HQ may cause a leukemogenic initiating event in a myeloblast such as a one of the translocations or deletions characteristic of acute myeloid leukemia [4]. At the same time it may covalently bind to the LTD4receptor to constitutively activate this signal-driven process and induce granulocytic differentiation which is incomplete and arrested at the myelocyte stage. Blocks in the developmental program of terminal hematopoietic cell differentiation appear to be a major step in tumor progression [34]. Concomitantly, BZ might provide a promotional effect on the initiated myeloblast via constitutive activation of protein kinase C causing overexpression of its activity with resultant pleiotropic effects on morphology and growth control. Indeed, such effects and susceptibility to malignant transformation have been observed when protein kinase C is overexpressed in fibroblast cell lines [35, 361, and increased protein kinase C activity is seen during induction of HL-60 myeloblasts to granulocytes by retinoic acid or dimethyl sulfoxide [37]. A recent report indicates that BZ, possibly by activation of protein kinase C, causes the hyperphosphorylation of p53, the growth suppressor that regulates entry into the cell cycle at the G, phase, and suggests that i Hazel/O'Connor/Niculescu/Kalf tumor promotion by BZ may involve hyperphosphorylation of p 5 3 [38]. Acknowledgments We are indebted to Carl Carlson for assistance with the experiments involving protein kinase C. 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