Document 7km5w1nRLGqJwzDwyvx2kvxj

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 O'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* LTD4 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 differentia tion of myeloblasts. BZ or HQ administered to C57BL/6J mice specifically induced terminal gran ulocytic differentiation of myeloblasts. The ability of the compounds to induce differentiation of the myeloblast was tested directly using the murine interleukin3 (IL-3)-dependent myeloblasticasU line, 32D.3 (G) and the human HL-60 promyelocytic leukemic cell line. Treatment of HL-60 myeloblasts with BZ activated protein kinase C and upregulated the 5-lipoxygenase (LPO) pathway for the produc tion of leukotrlene D< (LTD*), an essential effector of granulocytic differentiation. Differentiation was prevented by sphinganine, a kinase C inhibitor, as well as by LPO inhibitors and LTD* receptor antag onists. BZ and HQ also induced differentiation in 32D.3 (G) myeloblasts. Both compounds interact with cellular signaling pathways activated by gran ulocyte 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 growth in the absence of IL-3, but provides a differentiation signal. Both HQ and LTD* induce 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 pro gression of progenitor differentiation to granulo cytes 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 Sciences Building, Room 309,233 South Tenth Street, Philadelphia, PA 19107, USA. Received November 28, 1994; provisionally accepted February 16, 1995; accepted for publica tion February 28, 1995. AlphaMed Press 1066 5099/95/$5.00/0 STEM CHIB 1995;13:295-310 blocks differentiation at the myelocyte stage; only a small percentage of progenitors proceed to granu locytes. BZ, like G-CSF, upregulates LTD* produc tion, whereas HQ obviates the requirementfor LTD* by activating the LTD* receptor. Introduction Benzene (BZ), a widely used industrial chemical and ubiquitous environmental pollu tant, 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], phe nol, catechol and hydroquinone (HQ), are trans ported to the bone marrow [7,8] and further oxidized in a peroxidase-mediated [9-11] reac tion 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 deter mine whether BZ and/or a metabolite, such as HQ, directly affect the stem cell and/or progeni tor cells of the myeloid lineage. The ability to alter cytokine-dependent growth and differenti ation in hematopoietic progenitor cells appears to be a property of agents with leukemogenic poten tial for humans [12], There have been several reports on the effects of BZ on hematopoietic stem and progenitor cells [13], In one study [14], a dose-dependent depression of all stem cell com partments was observed in BDF1 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 of BZ. Dempster and C.A. Snyder [15] reported that short-term exposure of mice to BZ induced a shift toward granulocytic differentiation, and a growth advantage for granulocytic progenitor cells in the marrow and spleen which increased the total number of granulocytes. These results suggest that BZ and/or HQ is acting on the myeloid stem or progenitor cells. We report here that the administration of BZ or HQ specifically stimulates granulopoiesis in mice and induces granulocytic differentiation in myeloblasts of the human promyelocytic leukemic cell line, HL-60, and the normal murine interleukin 3 (IL-3)dependent myeloblastic cell line, 32D.3 (G). They do this by replacing the requirement for G-CSF and leukotriene D4 (LTD4), respectively, for induction of differentiation. Materials and Methods Animals C57BL/6J 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 facil ity (73" 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 accli mated 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. fetal bovine serum (FBS) .Cells used in the exper iments were from passages 18-42.The tL-3-depen dent myeloblastic cell line, 32D.3, was derived from normal bone marrow of C3H/HeJ mice by Greenberger[16]. It has a normal karyotype and is nonleukemic [16], The clone was further charac terized for IT-3 dependence by Metcalf [ 17], and for G-CSF induction of differentiation by Valtieri et al. [18] who gave the clone the designation (G). We are indebted to Dr. Giovanni Rovera for the kind gift of the 32D.3 (G) clone. The cells were maintained in Iscove's modified Dulbecco's medium (IMDM) in the presence of 10% FBS and 3 u/ml recombinant murine IT-3 (rMuIL-3) and supplemented with peni cillin (50IU/ml) and streptomycin (50mg/ml). Cells were cultured at 37Cin a 5%C02 atmos phere with biweekly replacement of medium and adjustment of cell concentration to 105 cells/ml for optimal growth. All cell cultures were demonstrated to be free of mycoplasma contamination by periodi cally testing the culture supernatant with a radiolabeled mycoplasma cDNA probe. Reagents IMDM,RPMI 1640, Dulbecco's phosphate buffered saline (PBS) minus Ca2+ and Mg!t, Tyrodes buffered salt solution (TBBS) and Pen/Strep were obtained from Mediatech, Washington, D.C. BZ (spectroanalyzed) and HQ were purchased from Fisher Scientific Co., Pittsburgh, PA. FBS, the AS-D Chloroacetate Esterase Assay Kit, LTD4, 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-dihydro-5-isoxazole acetic acid) were obtained from Sigma Chemical Co., St. Touis, MO. The 5-lipoxygenase inhibitor, AA861, was a product of Waco 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 Diego, CA was the source of the Mycoplasma Test Kit. 32Pi was a product of Dupont NEN, Boston, MA. Exposure of Cells to HQ, BZ, G-CSF, LTD4 and Other Agents HL-60 cells (5 x lOVml) were incubated with or without BZ in RPMI 1640/10% FBS for 7 days at 37 C after which differentiation to gran ulocytes was assessed. 32D cells (2.5 x 105/ml) Hazel/O'Connor/Niculescu/Kalf were incubated in IMDM/10% 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 glu cose (PBS-A) for 30 min at 37C after which the cells were collected, washed twice with PBS and placed into culture in IMDM/10% FBS with or without 3 u/ml rIL-3. G-CSF and LTD, were added directly to the culture medium. The lipoxygenase inhibitors and LTD4 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 105 cells/ml to insure optimal growing condi tions. Unless indicated otherwise in the fig ure 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 morphol ogy, specifically promyelocytes, metamyelocytes and segmented or mature cells, 2) the develop ment 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 differenti ation was performed by cytospin preparation and May Grtinwald/Giemsa staining. Per centages were based on the average obtained by counting 200 cells on each of triplicate slides. For NBT reduction, cells were incu bated for 1 hour with 0.125 nmol/1 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 chloroac etate and Red Violet at PH 6.3 for 5 min at 37*C, after which the percentage of cells con taining red granules was determined. The pres ence 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 (600 mg/kg 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 hema tology. 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 col lected in a tissue culture tube and a single cell suspension was prepared by passing the marrow through the syringe two additional times. A sam ple (100 pi) 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-HCl, pH 7.4, 155 mmol/1 NH4C1) 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 II) 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 lOOx. Five 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 HT-60 cells (3x lC^/ml) were twice washed in TBBS-Ca2t 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 4 $4 298 Induction of Granulopoiesis by Benzene and Hydroquinone in calcium and phosphate-free TBBS. The cells were washed three times with TBBS and incu bated 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 pi of a solu tion 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 [19], 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 carried out at 150 V for 3.5 h. The gel was silverstained, dried and exposed to X-OMAT AR film for 96 hat -70*C. HPLC Analysis of LTD4 Formation in HL-60 Cells Cells (1.5 X lOVml 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 100 pi 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 pi of mobile phase (65:35:0.02 v/v/v methanol:water:acetlc acid, pH 5.6) and 50 pi was injected into a reverse phase, 5 mm ODS HPLC column. An LTD4 standard (1 pg) was run as a control. Statistical Analysis Data between groups were analyzed using the t-testor ANOVA followedby Dunnett's t-test. Results are expressed as mean values SD. A p < 0.01 was considered significant. Results Stimulation of Granulocytic Differentiation in Mice by BZ and HQ As can be seen in Figure 1, the administra tion of BZ to mice stimulated the differentiation of myeloblasts as measured by an increased per centage of promyelocytes and intermediate gran ulocytic 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 4. CONTROL |BZ ho SO 20- ' 10- ' BLAST PM GB Fig. 1. The induction of granulocytic differentiation inC57BL/6J mice by BZ or HQ. Groups of mice (n = 4) were injected with BZ (600 mg/kg body weight) or HQ (25 or 50 mg/kg body weight) twice daily for 2 days. The control animals received com oil and PBSA. Eighteen h after the final injection the mice were killed, the bone marrow obtained and a morphologi cal analysis of the bone marrow carried out as described under Methods. Data are presented as absolute numbers of myeloid cells per femur repre sented 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 SD (n = 4); *p <0.001 as compared with the control group. The data from var ious cell types from the individual marrows are extremely tight (they range from SD 0.001 to 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 repre sentative 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 mg/kg body weight also stimulated granulocytic differentiation as indi cated 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 dif ferentiation of intermediate progenitors (band forms) to mature (segmented) granulocytes was Hazel/O'Connor/Niculescu/Kalf 299 MORPHOLOGYY CHLORACETATE NOW-SPCCIPIC JSSe,BEgSk* ESTEflASE ESTEBASE Fig. 2 BZ-induced granulocytic differentiationof HL60 myeloblasts. Cells (5 x lOVml) 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 charac teristics presented in the figure. The values represent the mean SD of at least three experiments where each sample was carried out in triplicate. *Significantly different from the control cells at p< 0.001. limited. HQ administered at 25 mg/kg body weight caused a lesser, but significant stimulation of granulopoiesis (data not shown). At these doses of BZ or HQ ,there was no loss of animals, no overt signs of toxicity and the viability of the cells flushed from the femurs remained at greater than 98%. Induction cf Granulocytic Differentiation in Myeloblasts by BZ and H Q The results of the in vivo experiments sug gested that BZ per se, or by metabolism to HQ, is capable of inducing granulocytic differentiation. However, they do not indicate whether either compound causes induction directly or indirectly in vivo. To answer this question and to have a 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 been adapted to terminally differentiate to gran ulocytes 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 locytic differentiation measured by morphology (determination of the percent of cells classified as promyelocytes or higher granulocytic forms) and the development of several characteristics of the granulocytic phenotype. No significant induc tion of nonspecific esterase activity indicative of monocytes was detected nor was monocyte mor phology observed (Table I). The ability of BZ to induce granulocytic morphology as well as the appearance of other markers of the granulocytic phenotype was a function of the concentration of the BZ used over the range 0.1 mM to 5 mM (data not presented). As can be seen in Table I, the HL60 cell population consists of about 45% promye locytes and 35% myeloblasts. BZ caused a significant decrease in the number of myeloblasts as well as promyelocytes, and a corresponding shift into more intermediate progenitors and mature granulocytes. The majority of the differ entiated progenitors appeared to be metamyelo cytes and myelocytes, but a significant number Table I. Morphological assessment of benzene-induced granulocytic differentiation of HL-60 cells System Blast Cell Type (%) Promyelocyte Myelocyte Metamyelocyte "Mature" Granulocyte Total 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-Griinwald/Giemsa stain. Percentages were based on the average obtained by counting 200 cells on each of triplicate slides in six different experiments. If a .V lW C t M W BZ-lnduced Activation cf 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 dif ferentiation of HL-60 myeloblasts to granulo cytes 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 32Pj to label ATP and treated with 5 mM BZ under con ditions 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 autoradi ography (Fig. 3). BZ induced the transfer of 32P| from AT[32P] to a number of HL-60 proteins (lane a) that was prevented by preincubation of HL-60 cells with 40 p M sphinganine prior to BZ treat ment (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 III-S (data not shown). Sphinganine also prevented BZ-induced granu locytic differentiation (Fig. 4), but by itself did not affect differentiation (data not shown). Since both BZ-induced phosphorylation of cellular pro teins and granulocytic differentiation were inhib ited 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 cf the 5-Lipoxygenase (LPO)Pathway in BZ-Induced Granulocytic Differentiation An active LPO pathway of metabolism that converts arachidonic acid to the peptidoleukotriene,LTD4l is essential for normal [23,24] as well as leukemic [25, 26] myeloid progenitor Fig. 3. Autoradiograph of an SDS-polyacrylamide gel separation of 32P-labeled cellular proteins from BZ-treated HL-60 cells. Cells (3 x 106/ml) were pre loaded with 3:P, 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.  CONTROL H BENZENE H BENZENE6 SPHINGANINE UntreatedHL-60 Celts REDUCTION Fig. 4. Inhibition of BZ-induced granulocytic differ entiation in HL-60 cells by sphinganine. Cells (5 x 1 O'/ml) were preincubated with 40 pM sphinganine in RPMI1640/10% FBS for 10 min at 37`C, 5% C02, The cells were exposed to 5 mM BZ and assessed for granulocytic differentiation after incubation for seven days as described under Methods.The values repre sent the mean SD cf at least three experiments where each sample was carried out in triplicate. *Significantly different from the control cells at p 10.0 01. cell proliferation and differentiation; LTD4 is an intermediate in CSF-induced clonal growth of GM-CFU [23, 24], The ability of BZ to cause the production of LTD4 and its immediate pre cursor, LTC4, under conditions where BZ induced granulocytic differentiation in HL-60 cells was investigated. A lipid extract of the culture medium of differentiating HL-60 cells grown in the presence and absence of a 5-LPO inhibitor was subjected to reverse-phase HPLC. In sev eral experiments, TTD, was produced in BZtreated cells in amounts two- to four-fold greater than in untreated cells. Figure 5 presents an HPTC profile from a typical experiment in which BZ induced the formation of LTD4 (two fold increase over untreated cells) in HL-60 cells. LTD4 is present in the untreated HL-60 cell pop ulation (Fig. 5) because approximately half of the cells are promyelocytes that have aborted their differentiation at that stage and thus con tain LTD4. The remainder are myeloblasts in which BZ should induce LTD, formation. The requirement for BZ-induced formation of LTD4 for granulocytic differentiation was demonstrated by showing that 5-LPO inhibitors prevented both BZ-induced LTD4 production and granulocytic differentiation in HL-60 myeloblasts (Table II), The highly specific 5-LPO inhibitor, AA-861 [27], prevented the formation of LTD4 induced in BZ-Trested BZ + LPO Inhibitor Fig. 5. BZ-induced formation of LTD, in HL-60 cells. Cells (1.5 x KP/ml in PBS) were incubated with or without 5 mM BZ (HPLC profiles 2 and 3) or with BZ and 1 pM AA861 (profile 4) for 24 h at 37'C. After removal of the cells, a 100 pi sample of the incubation medium was extracted with an equal volume of toluene and the organic phase evaporated to dryness. The residue was subjected to HPLC analysis for the presence of LTD, as described under Methods. Retention times (min) are printed above the peaks. An LTD4 standard (1 pg) was added as a control (profile 1). myeloblasts by BZ and stopped its constitutive production in the promyelocytes (Fig. 5). AA861 and another inhibitor, caffeic acid, prevented BZ-induced granulocytic differentiation (Table II), The inhibition was reversed by the addition of LTD4 concomitantly with the inhibitor. A highly specific LTD4 receptor antagonist, MK-571, [28] also completely inhibited BZ-induced granulo cytic differentiation in HL-60 myeloblasts, and this inhibition was prevented by the concomi tant addition of LTD4 (Fig. 6). Similar results (data not shown) were obtained with another receptor antagonist, LY 163443 [29], The conversion of the immediate precursor, LTC4, to LTD4 requires the removal of a glutamyl group from LTC4 by y-glutamyl transpeptidase [30], a reaction that is inhibited by the glutamine antagonist acivicin [23], Data presented in Table IE show that 1 pM acivicin prevented BZ-induced differentiation to granulocytes.This inhibition could be overcome by the addition of LTD4 to the culture medium, further supporting a role for LTD4 in BZ-induced differentiation of HL-60 cells to granulocytes. Taken together, these results suggest that BZ, via activation of protein 302 Induction of Granulopoiesis by Benzene and Hydroquinone Table II, Effect of 5-lipoxygenase inhibitors on benzene-induced granulopoiesis System MorphologyNTB Reduction Percentage of cells counted Control plus BZ plus BZ/caffeic acid plus BZ/caffeic acid/LTD4 plus BZ/AA861 plus BZ/AA861 /LTD4 12.3 5.5 37.5 3.8" 17.5 3.9 37.1 3-7b 23.7 3.3 54.7 10.5b 16.7 3.5 45.7 3.3" 20.8 3.7 42.8 8.3b 18.3 1.7 28.7 4.1b Cells (5 x lOVml) were preincubated in culture with5-lipoxygenase inhibitors caffeic add(100 pM), AA861 (1 pm) and with LTD4 (1 pM) in RPMI1640/15% FBS for 10 min at37C, 5%C02. BZ (5 nM) was added direcdy to the culture and NBT reduction and granulocytic differentiation were determined after incubation for seven days. Values represent the mean 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 0,01 kinase C, induces the formation of LTD4 required for the initiation of granulocytic differentiation. Induction of Granulocytic Differentiation in the Myeloblastic Cell Line 320.3 (G) by BZ and H Q In order to compare the induction of gran ulocytic differentiation by BZ and HQ with that of the normal physiological inducer, G-CSF, we turned to the IL-3-dependent, NBT REDUCTION MORPHOLOGY 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 [18], Table IV presents data from a representative of four experiments which gave similar results,There are few dif ferentiated 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 differenti ation 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 obvi ate the dependence of the cells on IL-3 for survival and growth. The Ability of LTD4 to Induce Granulocytic Fig. 6. Inhibition of BZ-induced granulocytic differ entiation in HF-60 cells by a 5-LPO inhibitor. Cells Differentiation in 32D Myeloblasts Since LTD4 is a downstream effector for i (5 x 1 CP/ml) were preincubated with 1 pM MK-571 in REMI1640/10% FBS orl0 min at37`C, 5%C02. BZ (5 mM) was added and granulocytic morphology G-CSF-induced signal transduction, and BZ has been shown to produce LTD4 via activation of protein kinase C and thus arachidonic acid was assessed after incubation for seven days. Values release, we tested the ability of LTD, to replace represent the mean SD of two experiments where G-CSF, BZ and HQ in the induction of granulo each sample was carried out in triplicate. *Significant cytic differentiation in 32D myeloblasts. As can I difference from the control cells at the p < 0.01 level; be seen in Figure 7, LTD4 is capable of replacing **significant difference from cells treated with BZ these inducing agents in a concentration-depen < and MK-57 1, but no significant difference from cells dent induction of terminal differentiation in 32D f treated with BZ only p < 0.01. myeloblasts. r Hazel/O'Connor/Niculescu/Kalf 303 Table ED, Effect of the y-glutamyl transpeptidase inhibitor, acividn cn benzene-induced granulocytic differentiation System Morphology NTB Reduction Percentage of cells counted Control plus BZ plus BZ/acivicin plus BZ/acivicin/LTD4 12.3 5.5 37.5 3.8* 22.0 1.0 45.3 11,9b 16.7 3.5 45.7 3.3' 23.5 4.2 47.3 9.1" Cells (5 x lOVml) were preincubated in culture with acivicin (1 pM) and LTD4 in RPMI1640/15% FBSfor 10 min at 37C, 5% C02, BZ (5 mM) was added direcdy to the culture and NBT reduction and granulocytic differentiation were determined after incubation for seven days. Values represent the mean SD of at least two experiments car ried out in triplicate. "Significantly different from control and BZ-treated cells pretreated with acivicin. ''Significantly different from cells treated with BZ and acivicin.p < 0.001. I1 Effects of Inhibitors of LTD4 Formation and of LTD4 Binding to Its Receptor on the Ability of HQ to Induce Granulocytic Differentiation in 320 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), 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 3.2 30.8+ 2.5 53.0 1.0 67.8 3.3 4.3 0.6 36.7 1.7 55.0 0.7 66.8 1.4 'Cells (2.5 x lOVml) were pretreated with HQ (2 pM in PBS-A) or PBS-A for 30 min at 37C.The cells were harvested ty centrifugation, suspended in IMDM (2.5 x 10s cells/ml) containing3 u/ml rMuIL-3 and 10%FB$. rG-CSF (0.15ng/ml) or BZ (5 mM) were added to the PBS-A-pretreated cells. Controls and HQ-pretreated cells received only PBS-A. After thrss days of culture at 37C, 5% C02, the medium was changed and the cells diluted to the original concen tration 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 SD of the results in triplicate wells. Data is pre sented as the percentage of cells showing granulo cytic differentiationout of a total of 200 cells counted. 'Includes cells from promyelocytic progenitors to termi nal 1 y differentiated granulocytes. causes the release of arachidonic acid, the sub strate for 5-TPO, 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 func tions in some other way, perhaps by interacting with the LTD4 receptor. An experiment was car ried out to ascertain whether the ability of HQ to induce terminal granulocytic differentiation in 32D myeloblasts could be prevented by an LTD4 receptor antagonist. The addition of the specific antagonist, MK571, completely blocked HQinduced 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 32D 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 prog enitor cell population is observed. As can be seen in Figure 8A, BZ does not provide a growth sig nal 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 IF-3 (Fig. 8A). Neither LTD4 nor HQ can induce a signal for prolifera tion in 32D myeloblasts in the absence of IL-3 (Fig. 8B). In the presence of IF-3, both LTD4 and HQ showed a significant stimulation of cell I 0 10-" lO* 107 10" LTD, CONCENTRATION (M) Fig. 7. LTD4-induced granulocytic differentiation in 32D myeloblasts. Cells (2.5 x 107ml) in IMDM (3 u/ml rIL-3; 10%FBS) were incubated with or without LTD4 for three days at 37C, 5% C02. Cells were thinned to the original cell number/ml and incubated an additional 3 days without the further addition of LTD4. Cytospin slides were stained for morphological analysis using May-Griinwald/Giemsa stain. Cells were monitored i* for superoxide production by following NBT reduc tion using TPA/NBT and counterstaining with Safranin. 12 3 DAYS POST TREATMENT growth which, in the case of HQ, was more than 100%at day 4. A Comparison cf the Kinetics cf Stage-Specific Granulocytic Differentiationlnduced by the Physiological Inducers G-CSF and LTD,, and by BZ and HQ These agents all transmit a signal for dif ferentiation 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 pro liferation 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 com petes 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 con trast to BZ, HQ or LTD4, can provide signals for proliferation and differentiation. Conse quently, 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 107ml) were incubated in IMDM/10% FBS with 5 mM BZ or rG-CSF (500 u/ml) with or without rIL-3 (3 u/ml) for eight days at 37"C, 5% COi, B) Cells were pretreated with 2 pM HQ/PBS-A or PBS-A for 30 min at 37"C, 5% C02, washed twice with PBS-A. PBS-A-treated cells were also treated with LTD4 (3.8 pM). All 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 sam ples 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 ver sus terminal differentiation on day 6 in the absence of IL-3) is presented in Figure 9. G-CSF induced predominantly, terminally differenti ated granulocytes whereas HQ induced pre dominantly myelocytes. BZ and LTD4 produced a significant number of terminally differenti ated granulocytes as well as an increased num ber 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 LTD4 receptor antagonist (MK571) on G-CSF- and HQ-induced differentiation of 32D cells System MorphologyNTB Reduction Percentage of cells counted A. Control +G-CSF +G-CSF + 5-TPO inhibitor +HQ +HQ + 5-LPO inhibitor 17.5 2.6 70.8 3.5 31.9 0.9 81.5 1.3 66.1 1.7 12.5 3.6 48.2 10.6 12.6 2.4 48.6 5.7 38.6 2.0 B. Control +G-CSF +G-CSF + R antagonist +HQ +HQ + R antagonist 17.5 2.6 70.8 3.5 32.8 8.8 81.5 1.3 24.1 1.8 12.5 3.6 48.2 10.6 26.8 6.2 48.6 5.7 15.2 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-TPO inhibitor, AA861, or the LTD4 receptor antagonist, MK571, at 1 p M final concentration. Morphological analysis was performed as described in the legend for Table IV. 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 granulo cytic 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 compara ble 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 Day 0 Percent of Total Differentiation Day 2 Day 4 Day 6 Percent of Terminal Differentiation Day 6 IL-3 IL-3 + G-CSF G-CSF IL-3 + BZ BZ IL-3 + LTD4 LTD4 IL-3 +HQ HQ 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.0 2.0 0.0 19.3 0.4 3.3 0.3 19.0 2.2 9.2 3.2 32.0 0.0 4.3 1.4 40.0 0.5 0.7 0.3 24.0 2.0 71.5 0.9 22.3 1.6 89.8 1.1 30.2 2.8 97.0 0.5 41.8 2.3 91.5 0.0 6.0 0.0 91.8 1.6 90.8 1.6 86.8 2.3 99.7 0.6 92.8 1.3 99.3 0.3 88.5 2.8 98.8 0.3 1.0 16.7 51.2 18.0 39.8 25.2 46.8 14.8 17.2 Cells (2.5 x lOVml) were treated as describedin 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 segmented/banded granulocytes over the total number of cells counted for each sample on day 6. day 8, almost all of the early progenitor forms had terminally differentiated to granulocytes. In comparison, LTD4, 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 six- day period (Fig. 10B), There were many more intermediate progenitors (myelocytes) and ter minally differentiated granulocytes at day 2 in the presence of exogenous LTD4) most probably because the cells do not have to wait for LTD4 to be produced following the G-CSF signal. At Fig. 9. Granulocytic differentiation in 32D cells 6 day 4, the promyelocyte burst seen with G-CSF is days post-treatment with inducing agents. Cells (2.5 absent and the percentage of intermediate and x 105/ml) were treated with inducing agents as mature forms (45%) is significantly increased described in the legend for Figure 8. Samples of each because the effector was added directly and there culture were taken on day 6 and Cytospin slides were was no proliferation signal. By day 6, the mye j prepared in triplicate for each culture and stained for locytes have begun to mature into metamyelo morphological analysis with May-Grtinwald7 Giemsa cytes and the number of terminally differentiated as described under Methods. Two hundred cells were counted and an average percent for each stage of dif granulocytes has remained constant, probably * ferentiation was determined. PM, promyelocytes; because those that were mature at day 4 are \i. MY, myelocytes; ME, metamyelocytes; MA, mature beginning to die off. Because of the lack of a segmentedhanded granulocytes. proliferation signal and the rapid induction of |3 ot- & PMyMM, PMyMM, PMyMM, PMyMM, 5oHI LTD4-TREATED jB jjj J PMyMM, PMyMM, PMyMM, PMyMM, DAYP DAY4 DAY6 DAY8 o PMyMM. PMyMM, PMyMM. PMyMM. Fig. 10. Profile of stage-specific differentiation induced by G-CSF (A), LTD4 (B), BZ (C) and HQ (D). Cells (2.5 i x lOVml) were treated as described in the legend for Figure 8. Samples of 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 calcu lated by totaling the percentages of stage-specific differentiationfor each sample. P, promyelocyte; My, myelocyte; I M, metamyelocyte; Ma, mature granulocyte. Hazel/O'Connor/Niculescu/Kalf 307 differentiation in the presence of LTD4, there were few viable cells by day 8; however, those that remained were 100% terminally differenti ated (data not shown). Thus, while the pattern of differentiation with G-CSF or LTD4 was dif ferent, they had in common the fact that at each day, the major progenitor type was the termi nally differentiated granulocyte. BZ induced a pattern of differentiation at day 2 that was qual itatively similar to that seen with G-CSF or LTD4 (Fig. 10C). At day 4, BZ showed a pattern cf differentiation similar to LTD4 except that the predominant progenitor type was the myelocyte rather than the terminally differentiated granu locyte. At day 6 the BZ-induced pattern contin ues 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 granulo cytic differentiation in 32D myeloblasts beyond the myelocyte stage at day 4 which resolves itself at day 6 (Fig. 10C), whereas HQ appears unable to induce significant differentiation beyond the myelocyte stage (Fig. 10D). Here, as in the case of LTD4-induced differentiation, the number of viable cells present at day 8 was very low, but those present were mature granu locytes in the case of BZ and with HQ, a mixture of predominantly intermediate progenitors and some mature granulocytes. Discussion Exposure of C57BL/6J mice to a dose of BZ known to depress other hematopoietic cell lin eages [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 inter mediate progenitors. It did not stimulate the pro duction of mature granulocytes at day 3, however, when measured at day 7, it had increased the number of segmented, mature gran ulocytes (data not shown). The results showing a general increase in granulocytes after BZ expo sure are consistent with those showing an increase in the number of granulocytes in bone marrow of DB A72J mice after short-term expo sure to BZ [15], HQ, a major metabolite of BZ found in the bone marrow, also induced growth and differentiation of granulocytic progenitor cells measured at day 3 when administered to C57BL/6J mice (Fig. 1) as well as in mouse 32D myeloblasts in culture (Fig. 8B). In contrast to BZ, HQ pro vided both growth and differentiative signals for myeloblasts that increased the numbers of all prog enitor 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 inves tigated 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-depen dent specific induction of terminal granulocytic differentiation in HL-60 myeloblasts (Table I, Fig. 2), as measured by the determination of granulocytic morphology and functional para meters of granulocytic differentiation such as superoxide production, chloroacetate esterase activity and the appearance cf the specific sur face antigen, L- 12-2. BZ induction produced a majority of intermediate progenitors (metamye locytes 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 dif ferentiation. 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 spe cific 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 LTD4 since the differentia tion 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 M A U R IC E A N D L A U R A fA U ' 308 the LTC4 step by acivicin, an inhibitor of y-glutamyl transpeptidase (Table 111). Inhibition by all of these agents was prevented by the con comitant addition of LTD4 with the agent. These results together support the view that the role of BZ in inducing granulocytic differ entiation in HL-60 myeloblasts is to produce LTD* that is a necessary, if not a sufficient, sig nal for granulocytic differentiation. BZ appears to function in a manner similar to the physio logical 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 phos pholipase A2 and 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 (GjUerived 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 differ entiation 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 dif ferentiation. 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 Ironsef 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, sig nificantly 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 dif ferentiation in myeloblasts in culture is pre vented 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 granu lopoietic activity of BZ in vivo can be attrib uted to its metabolism to HQ. During chronic BZ exposure, BZ and HQ are present in the bone marrow and consequently both can con tribute 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 pro duction of LTD4. HQ appears to obviate the requirement for LTD4 by activating the LTD4 receptor since specific LTD4 receptor antago nists, 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 LTD4 receptor 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 termi nal hematopoietic cell differentiation appear to be a major step in tumor progression [34], Con comitantly, BZ might provide a promotional effect on the initiated myeloblast via constitutive activation of protein kinase C causing overex pression of its activity with resultant pleiotropic effects on morphology and growth control. Indeed, such effects and susceptibility to malig nant transformation have been observed when protein kinase C is overexpressed in fibroblast cell lines [35, 36], and increased protein kinase C activity is seen during induction of HL-60 myeloblasts to granulocytes by retinoic acid or dimethyl sulfoxide [37], A recentreport indicates that BZ, possibly by activation of protein kinase C, causes the hyperphosphorylation of ^53, the growth suppressor that regulates entry into the cell cycle at the G| phase, and suggests that HazeL/O'Connor/Niculescu/Kalf tumor promotion by BZ may involve hyper phosphorylation ofp53 [38], Acknowledgments We are indebted to Carl Carlson for assis tance with the experiments involving protein kinase C. This research was supported, in part, by EPA grant R8 19301. Betsy Hazel is supported by a Graduate Stipend from the American Petroleum Institute. References 1 Aksoy M. Malignancies due to occupational expo sure to benzene. 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