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Hydroquinone, a Bioreactive Metabolite of Benzene, Inhibits Apoptosis in Myeloblasts

Department of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

Key Words. Hydroquinone • Benzene metabolite • Myeloblasts • Inhibition of apoptosis

Dr. George F. Kalf, Department of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Thomas Jefferson University, M-5 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Hydroquinone (a major marrow metabolite of the leukemogen, benzene) induces incomplete granulocytic differentiation of mouse myeloblasts to the myelocyte stage, and also causes an increase in the number of myelocytes. This was confirmed using the normal interleukin 3 (IL-3)-dependent mouse myeloblastic 32D cell line. The hydroquinone-induced twofold increase in the number of IL-3-treated myelocytes does not result from stimulation of IL-3-induced proliferation. Hydroquinone's ability to effect this increase through an inhibition of apoptosis was investigated using mouse 32D and human HL-60 myeloblasts. Apoptosis induced by staurosporine treatment (0.5-1.0 µM) of HL-60 cells (50%) and 32D cells (15%) or by IL-3 withdrawal from 32D myeloblasts was determined by monitoring the development of characteristic morphological features and confirmed by the appearance of a typical nucleosomal DNA ladder upon agarose gel electrophoresis. Concentration of hydroquinone (1-6 µM) that induce differentiation in 32D myeloblasts caused a concentration-dependent inhibition of staurosporine-induced apoptosis in both cell lines, with a 50% inhibitory concentration of 3 µM, and prevented apoptosis in IL-3-deprived 32D cells. Hydroquinone inhibition of apoptosis in myeloblasts, like hydroquinone-induced granulocytic differentiation, required myeloperoxidase-mediated oxidation of hydroquinone to its reactive species, p-benzoquinone, and was inhibited 50% by the peroxidase inhibitor, indomethacin (20 µM). p-benzoquinone (3 µM) was shown to cause a 50% inhibition of CPP32, an IL-1ß converting enzyme/Ced-3 cysteine protease involved in the implementation of apoptosis and present in myeloid cells. The ability of hydroquinone to induce a program of differentiation in the myeloblast that proceeds only to the myelocyte stage coupled with its ability to inhibit the CPP32 protease and, thereby, apoptosis of the proliferating myelocytes, may have important implications for benzene-induced acute myeloid leukemia.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Benzene (BZ), a widely used industrial chemical and ubiquitous environmental pollutant, is a class I carcinogen that causes acute myeloid leukemia (AML) in humans that are chronically exposed [1-4]. Hydroquinone (HQ), a major metabolite of BZ found in the bone marrow [5-6], is converted, in myeloid cells, in a myeloperoxidase-mediated reaction [7-9] to bioreactive p-benzoquinone (BQ) that reacts with sulfhydryl-dependent proteins [10-14] and other cellular macromolecules in ways that may affect hematopoiesis in general, and myelopoiesis in particular. We have reported that BZ or HQ induces granulopoiesis in mice [15] and causes granulocytic differentiation in myeloblasts of the normal murine, interleukin (IL)-3-dependent, G-CSF-inducible cell line, 32D cl 3(G) [15], and the human promyelocytic leukemia cell line, HL-60. BZ per se induces granulocytic differentiation by upregulating the production of leukotriene D₄ (LTD₄), an essential physiological downstream mediator of G-CSF-induced granulocytic differentiation in myeloblasts [15]. HQ causes a concentration-dependent induction granulocytic differentiation in 32D myeloblasts via oxidation to BQ which binds to the LTD₄ receptor. HQ (BQ) induces differentiation predominantly to the myelocyte stage, whereas binding of LTD₄, the physiological ligand, induces terminal granulocytic differentiation [15]. In the hematopoietic system, cytokines induce cell growth that is coupled to differentiation such that clonal expansion of a differentiating cell population is observed. G-CSF is capable of inducing both growth (measured as increased cell number) and terminal granulocytic differentiation in IL-3-dependent 32D myeloblasts in the absence of IL-3. HQ prevents the loss of cells that normally occurs in the myeloblast population in the absence of IL-3, whereas in the presence of IL-3, HQ significantly increases the number of cells in the myeloblast/myelocyte population. These results suggest that HQ, in addition to including granulocytic differentiation in myeloblasts, causes the increase in the myelocyte population by affecting cell proliferation and/or programmed cell death (apoptosis) of myeloblasts. The ability to alter cytokine-dependent growth and differentiation of hematopoietic stem and/or progenitor cells appears to be a property of agents with leukemogenic potential for humans [16]. We present data which demonstrate that HQ, at a concentration that induces granulocytic differentiation, increases the number of myelocytes by inhibiting (delaying) apoptosis in cytokine (IL-3)-deprived mouse 32D myeloblasts and staurosporine (SP)-induced apoptosis in 32D and HL-60 myeloblasts.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Reagents
Iscove's modified Dulbecco's medium (IMDM), RPMI 1640, and Dulbecco's phosphate buffered saline (PBS) minus Ca²⁺ and Mg²⁺ were obtained from Mediatech (Washington, DC). HQ, indomethacin and fetal bovine serum (FBS), as well as May-Grünwald and modified Giemsa stains were purchased from Sigma Chemical Co. (St. Louis, MO). SP was obtained from the Kamiya Biochemical Co. (Thousand Oaks, CA). Recombinant murine (rMu) IL-3 was obtained from R&D Systems (Minneapolis, MN). DNAzol reagent and the 1 Kb DNA standard molecular weight ladder were purchased from Life Technologies (Gaithersburg, MD). Gen Probe (San Diego, CA) was the source of the Mycoplasma Test Kit.

Cell Culture
The normal IL-3-dependent murine myeloblastic cell line, 32D cl 3 G [17,18], was adapted for G-CSF induction of differentiation [19]. HL-60 human promyelocytic leukemia cells [20] were obtained from the American Type Culture Collection (ATCC #240-CCL). The cells were maintained and cultured as previously described [15]. 32D cells used in the experiments were from passages 4-20; HL-60 cells were from passages 18-42. Cell number was determined with a hemocytometer, and cell viability by trypan blue exclusion. Cultures were demonstrated to be free of mycoplasma contamination by periodic testing of the culture supernatant with a radiolabeled mycoplasma cDNA probe.

Treatment of Myeloblasts with IL-3, HQ or SP
32D myeloblasts (2.5 or 5.0 x 10⁵/ml) were incubated in IMDM/10% FBS/2 mM glutamine, with or without rMuIL-3 and/or the agent to be tested. When HQ was used, the cells were either pretreated with HQ in PBS/2 mM glucose (PBS-A) for 30 min or 1 h at 37°C, after which the cells were collected and placed into culture in IMDM/10% FBS/2 mM glutamine with or without rMuIL-3, or the HQ was added directly to the culture medium. In the latter case, twice the concentration of HQ was required to cause a comparable result due to the propensity of HQ to interact with proteins in the medium. After the appropriate period of incubation, a sample of each culture was removed for cell counting, Cytospin slide preparation, and May Grünwald/Giemsa staining. HL-60 cells (2.5 or 5.0 x 10⁵/ml) were incubated in RPMI 1640/20% FBS/2 mM glutamine and treated as the 32D cells were, with the exception that IL-3 was not required. The concentrations of agents used were not cytotoxic to the cell lines and the viability of the cells after treatment was >98% as measured by trypan blue exclusion.

Proliferation Assay
Proliferation was determined by measuring the incorporation of [³H] thymidine into the DNA of synchronized myeloblasts over a 48 h interval. 32D cells were synchronized by withdrawal from IL-3 for 16h, after which they were incubated in the presence or absence of HQ followed by suspension in IL-3 medium, as described above. At 0 h an aliquot (5 x 10⁴ cells) of each culture (in triplicate) was incubated in the presence of 0.5 µCi [³H] thymidine for 24 h, after which [³H] thymidine incorporation was measured. To ensure that HQ did not have a delayed effect on proliferation, a second aliquot of cells was removed from each of the original cultures at 24 h and incubated as above. Cells were collected on a filter, washed three times with ice cold 10% TCA, and once with ethanol. Air-dried filters were placed in a vial with 0.5 ml Protosol and 5 ml BCS scintillation fluid, and radioactivity was measured using a Beckman LS 3801 scintillation counter.

Assessment of Apoptosis
Apoptosis was assessed in HL-60 cells by ascertaining the acquisition of morphologic changes such as micronucleus formation. Morphologic assessment of apoptosis was carried out by averaging the number of cells showing apoptotic changes among 200 cells on each of triplicate slides. In the case of HL-60 cells, induction of apoptosis was confirmed by in situ detection using DAPI staining. The presence of apoptosis in 32D cells was indicated by micronuclei, surface blebbing, and the presence of apoptotic bodies. However, these changes occurred too rapidly to use as a quantitative marker in the assessment of apoptosis. Internucleosomal DNA fragmentation manifested by a DNA ladder upon gel electrophoresis was also determined for both cell lines as described below.

Staining of Cytospin Preparations
Slides were fixed in 95% ethanol for 1 min and allowed to air dry, after which they were stained with May-Grünwald stain for 5 min followed by a 3 min wash in cold PBS and counterstaining in Giemsa for 18 min. Slides were air-dried and examined for apoptotic morphology using oil immersion at 100x.

Gel Electrophoresis
DNA from cell pellets (2 x 10⁷ cells) was extracted with 1 ml DNAzol, purified by 2 ethanol precipitations, solubilized in 8 mM NaOH, and quantified spectrophotometrically. DNA (1-3 µg) was loaded into each well of a 1.5% agarose gel containing 1 µg/ml EtBr in Tris-acetate (TAE) buffer. An additional 1 µg/ml EtBr was added to the anode buffer pool in the electrophoresis chamber. Gels were run at 23°C at a constant voltage of 100 V for 1 h, 40 min. The presence of DNA ladders was visualized in comparison with a 1 Kb standard DNA Ladder by exposure of the EtBr-intercalated DNA to ultraviolet light. Photography of the gel was carried out with a Mitsubishi Camera/Video Processor (Mitsubishi Electric Corp., Japan).

Assay of Protease Activity of CPP32
Cloned CPP32 (1 µg) was incubated in IL-ß converting enzyme (ICE) assay buffer with 50 mM DEVD-AMC peptide substrate, with or without 2.5 µM BQ, in 100 µl of reaction mixture according to the method of Fernandez-Alnemri et al. [21]. The release of AMC (7-amino-4-methylcoumarin) was measured over a 30 min period by spectrofluorometry at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

Statistical Analysis
Data between groups were analyzed using Student's t-test. Results are expressed as mean values ± SD. Each experiment was repeated at least two times with similar or identical results. In figures where individual data points do not show error bars, the variation was too close to plot.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Growth Characteristics of 32D and HL-60 Myeloblasts
Human HL-60 tumor cells, which grow logarithmically in the presence of serum alone (Fig. 1A), show no evidence of cell death during the four-day test period as measured by trypan blue exclusion (Fig. 1B). In comparison, normal mouse 32D myeloblasts show increased growth in the presence of 3 U/ml rMuIL-3, which peaks between three and four days in culture, after which the cell number decreases due to a depletion of the cytokine (Fig. 1A). Passage number of the culture, IL-3 concentration, and cell number per ml all affect the precise timing of the growth peak (data not shown). The decrease in cell number correlates well with the observed loss of viability as measured by trypan blue exclusion (Fig. 1B). In the absence of IL-3, the number of myeloblasts (Fig. 1A) and their viability (Fig. 1B) decreases by 90% at day 2. Similar results have been reported [22, 23] to represent apoptosis in IL-3 dependent 32D myeloblasts. That apoptosis is occurring in our experiments under conditions of IL-3 deprivation can be seen by the decrease in the number of dividing cells and the progressive development of characteristic morphological features (Figs. 2D-2F) such as cytoplasmic condensation, cell volume reduction, cytoplasmic vacuolization and blebbing, chromatin condensation, densely stained micronuclei, and subsequent fragmentation of the cell into small membrane-bound vesicles or apoptotic bodies as compared with IL-3-treated cells (Figs. 2A-2C). These features were apparent as early as 6 h after IL-3 withdrawal (Fig. 2E) and became more pronounced with time (Fig. 2F) with the appearance of internucleosomal DNA fragments in 32D cells occurring as early as 6 h after removal of IL-3. While the culture that was incubated with IL-3 showed very little apoptotic morphology at 48 h (Fig. 2C), virtually the entire IL-3-deprived culture was composed of cells with highly condensed cytoplasm and chromatin, micronucleated cells, and/or apoptotic bodies (Fig. 2F). Chromatin condensation, which occurs early in apoptosis, is the result of double-stranded cleavage of DNA by endonucleases that leads to the formation of internucleosomal DNA fragments. The appearance of such fragments in 32D cells as early as 6 h after removal of IL-3 (Fig. 3), as monitored by the appearance of a DNA ladder upon gel electrophoresis of the DNA, correlated well with the morphological data (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Figure 1. Growth and viability of HL-60 and 32D myeloblasts. HL-60 cells (2.5 x 105/ml) were seeded in RPMI 1640 with 20% FBS/2 mM glutamine. 32D cells (2.5 x 105/ml) were seeded in IMDM with 10% FBS/2 mM glutamine in the presence or absence of 3 U/ml rMuIL-3. Cultures were incubated at 37°C for four days and samples were collected daily for the determination of growth and viability. Growth (A), measured as cell number/ml, was determined by hemacytometer. Viability (B) determined by trypan blue exclusion.

View larger version (149K): [in this window] [in a new window]   Figure 2. Effect of IL-3 withdrawal on 32D myeloblast morphology. 32D cells (5 x 105/ml) were incubated at 37°C for 48 h in the presence or absence of IL-3. At specified intervals, samples were collected (8 x 104 cells), spun down and suspended in 50% FBS/50% RPMI at a concentration of 2.5 x 105/ml. Cytospin slides were made in triplicate, stained with May-Grünwald/Giemsa, and examined under oil at 100 x . (A-C) with IL-3; (A) 0 h, (B) 6 h, (C) 48 h. (D-F) minus IL-3; (D) 0 h (E) 6 h, (F) 48 h. Arrows indicate dividing myeloblasts or apoptotic cells.

View larger version (110K): [in this window] [in a new window]   Figure 3. DNA fragmentation in 32D myeloblasts induced by IL-3 withdrawal. 32D cells (5 x 105/ml) were incubated at 37°C for 48 h in the presence or absence of IL-3. At specified intervals, samples were collected (1-2 x 107 cells) and DNA was extracted with DNAzol, precipitated by ethanol and solubilized in 8 mM NaOH. One microgram of DNA (by A260) was loaded per lane in a 1.5% agarose gel containing 1 µg/ml EtBr and electrophoresis was carried out in TAE buffer at 100 V for 1 h, 40 min.

View larger version (12K): [in this window] [in a new window]   Figure 4. Effect of HQ on growth of 32D myeloblasts in the presence or absence of IL-3. 32D cells (2.5 x 105/ml) were pretreated with 2 µM HQ/PBS-A, or PBS-A alone, for 30 min at 37°C, after which they were suspended in IMDM/10% FBS/2 mM glutamine in the presence or absence of 3 U/ml rMuIL-3. Cultures were incubated at 37° C for four days. Samples were collected every second day and analyzed for growth (cell number/ml) by hemocytometer.

View larger version (10K): [in this window] [in a new window]   Figure 5. Effect of HQ on proliferation of 32D cells in the presence of IL-3. 32D cells were synchronized by withdrawal from IL-3 for 16 h, after which they were incubated in the presence or absence of HQ, followed by suspension in IL-3 medium as described in Figure 4. At 0 h an aliquot (5 x 104 cells) of each culture (in triplicate) was incubated in the presence of 0.5 µCI [3H] thymidine for 24 h, after which [3H] thymidine incorporation was measured. A second aliquot of cells was removed from each of the original cultures at 24 h and incubated as above. Cells were collected on a filter, washed three times with ice-cold 10% TCA, and once with ethanol. Air-dried filters were placed in a vial with 0.5 ml Protosol and 5 ml BCS scinitillation fluid and radioactivity was measured using a Beckman LS 3801 scintillation counter.

View larger version (76K): [in this window] [in a new window]   Figure 6. Inhibition of DNA fragmentation in IL-3-deprived 32D myeloblasts by HQ. 32D myeloblasts (5 x 105/ml) were pretreated with 3 µM HQ/PBS-A, or PBS-A alone, for 1 h at 37°C, after which they were suspended in IMDM/10% FBS/2mM glutamine in the presence or absence of 3 U/ml rMuIL-3 and incubated for 18 h. Samples were collected (1-2 x 107 cells) and DNA was extracted with DNAzol, precipitated by ethanol and solubilized in 8 mM NaOH. Three micrograms of DNA (by A260) were loaded per lane in a 1.5% agarose gel containing 1 µg/ml EtBr and electrophoresis was carried out in TAE buffer at 100 V for 1 h, 40 min.

View larger version (11K): [in this window] [in a new window]   Figure 7. Inhibition of SP-induced apoptosis in 32D myeloblasts by HQ. 32D myeloblasts (5 x 105/ml) were pretreated with 3 µM HQ/PBS-A, or PBS-A alone, for 1 h at 37°C, after which they were suspended in IMDM/10% FBS/2 mM glutaimine/3 U/ml rMuIL-3 in the presence or absence of SP (1 µM). Cultures were incubated at 37°C for 12 h, after which samples were collected (8 x 104 cells), spun down and suspended in 50% FBS/50% IMDM at a concentration of 2.5 x 105/ml. Cytospin slides were made, stained with May-Grünwald/Giemsa, and examined under oil at 100 x . The percent apoptosis was calculated by determining the number of micronucleated cells out of 200 total cells counted on each of triplicate slides per sample. C: control, H: HQ, S: SP. *Significantly different from S sample at a level of p 0.01.

View larger version (12K): [in this window] [in a new window]   Figure 8. Concentration-dependent inhibition of SP-induced apoptosis in HL-60 cells by HQ. HL-60 cells (5 x 105/ml) were incubated at 37°C for 10 min in RPMI 1640/20% FBS/2 mM glutamine in the presence or absence of various concentrations of HQ. SP (0.5 µM) was then added to specified cultures, after which all cultures were incubated at 37°C for an additional 3 h. Following incubation, samples of each culture were collected (8 x 104 cells), spun down and suspended in 50% FBS/50% RPMI at a concentration of 2.5 x 105/ml. Cytospin slides were made and stained with May-Grünwald/Giemsa, and examined under oil at 100 x . The percent apoptosis was calculated by determining the number of micronucleated cells out of 200 total cells counted on each of the triplicate slides per sample. *Significantly different than SP sample at a level of p 0.001. **Significantly different than SP/1 µM HQ sample at a level of p 0.02.

View larger version (58K): [in this window] [in a new window]   Figure 9. Inhibition of DNA fragmentation in SP-treated HL-60 myeloblasts by HQ. HL-60 cells (5 x 105/ml) were pretreated with 3 µM HQ/PBS-A, or PBS-A alone, for 1 h at 37°C, after which they were suspended in RPMI/20% FBS/2 mM glutamine in the presence or absence of SP (0.5 µM) and allowed to incubate at 37° C for an additional 3 h. Cell pellets (1-2 x 107) were then collected and DNA was extracted with DNAzol, precipitated by ethanol, and solubilized in 8 mM NaOH. Three micrograms of DNA (by A260) were loaded per lane in a 1.5% agarose gel containing 1 µg/ml EtBr and electrophoresis was carried out in TAE buffer 100 V for 1 h, 40 min. Qualitatively similar results were seen when DNA was obtained from cells incubated under the conditions in A; however, a 1 h preincubation with HQ enhances the inhibition observed.

View larger version (13K): [in this window] [in a new window]   Figure 10. Effect of indomethacin on HQ's ability to inhibit SP-induced apoptosis in HL-60 myeloblasts. HL-60 cells (5 x 105/ml) were incubated at 37°C for 20 min in RPMI 1640/20% FBS/2 mM glutamine in the presence or absence of indomethacin (25 µM). HQ (6 µM) was then added to specified cultures, after which all cultures were incubated at 37°C for an additional 10 min. Finally, SP (0.5 µM) was added to the indicated cultures, and all cultures were incubated at 37°C for 3 h. Following incubation, samples of each culture were collected, spun down, and suspended in 50% FBS/50% RPMI at a concentration of 2.5 x 105/ml. Cytospin slides were made and stained with May-Grünwald/Giemsa. The percent apoptosis was calculated by determining the number of micronucleated cells out of 200 total cells counted on each of triplicate slides per sample. *Significantly different from SHI sample at a level of p 0.001.

A common feature of these proteases is conservation of a peptide sequence, QACRG, containing a cysteine sulfhydryl group at the active site. Since BQ avidly reacts with the cysteine-SH group, all of the proteases should be amenable to inhibition by BQ. We have previously demonstrated that calpain, a sulfhydryl-dependent protease required for the conversion of pre-IL 1 to IL-1 is inhibited by BQ [14, 50] and that BQ is covalently bound as the monoadduct of S-cysteine in a heptapeptide from the active site of calpain (unpublished experiments). BQ also inhibits the conversion of pre-IL-1ß to mature cytokine by ICE [50]. CPP32 is the cysteine protease present in myeloblasts/promyelocytes [21]. Consequently, an experiment was carried out to determine whether BQ inhibited the ability of CPP32 to hydrolyze the peptide substrate, DEVD-AMC. As can be seen in the representative kinetic experiment presented in Figure 11, 2.5 µM BQ caused a 50% inhibition (IC₅₀) of the protease activity of CPP32. This concentration of BQ was chosen not only because it is the IC₅₀ concentration for the inhibition of ICE, another CPP32 family member, but also because it represents the concentration which results in an increased cell number in the presence of IL-3, maximal differentiation and 50% inhibition of apoptosis.

View larger version (13K): [in this window] [in a new window]   Figure 11. Inhibition of the apoptotic cysteine protease, CPP32 by BQ. DEVD-AMC peptide substrate (50 µM) was incubated with cloned CPP32 (1 µg) in an ICE assay buffer with or without 2.5 µM BQ in a final volume of 100 µl according to the method of Fernandes-Alnemri et al.[21]. Protease activity was monitored by measuring the release of AMC (7-amino-4-methylcoumarin) over a 30 min period by spectrofluorometry at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Control buffer gave a value of 3.9 in both cases.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

SP, a protein kinase inhibitor which induces a common pathway of apoptosis in a number of cell lines from various origins [24], was used to determine whether HQ could prevent apoptosis induced by means other than cytokine withdrawal. Although it is difficult to induce apoptosis in normal 32D mouse myeloblasts with SP (Fig. 7) relative to that induced in HL-60 myeloblasts (Fig. 8) which overexpress myc [20, 25] HQ (3 µM) is capable of inhibiting SP-induced apoptosis in both cell lines (Figs. 7-9).

The ability of HQ to inhibit SP-induced apoptosis is inhibited by the peroxidase inhibitor, indomethacin, [9], and thus is dependent upon HQ's MPO-mediated oxidation to bioreactive BQ in myeloblasts (Fig. 10). BQ covalently reacts with sulfhydryl-dependent proteins [10-14, 50] to form the monoadduct of S-cysteine. We have previously demonstrated [50] that BQ inhibits the activity of the human sulfhydryl-dependent cysteine protease, ICE, a member of the ICE/Ced-3 family of proteases involved in the regulation of apoptosis [33-49]. The ability of BQ to inhibit ICE suggested a molecular mechanism whereby HQ might inhibit apoptosis in myeloblasts. BQ, at a concentration equivalent to that known to inhibit ICE [50], as well as both SP-induced (Fig. 9B) and cytokine-withdrawal-induced (Fig. 6) apoptosis in myeloblasts, also showed a 50% inhibition of the sulfhydryl-dependent protease activity of CPP32 (Fig. 11), the ICE/Ced-3 family member responsible for apoptosis in myeloblasts/promyelocytes [21]. Thus, BQ most probably prevents apoptosis in 32D myeloblasts because of its ability to interact with the cysteine SH group at the conserved QACRG active site of CPP32.

Chronic exposure of humans to BZ induces, almost exclusively, AML [1-4]. To date, no animal model has been developed which is sufficient for the study of BZ-induced leukemia. However, HQ, a major metabolite of BZ in the bone marrow, when administered to the mouse, induces granulopoiesis [15]. Similarly, HQ, via peroxidation to BQ in the normal committed myeloid stem cell represented by 32D cells in culture, induces granulopoiesis providing a useful model system for investigating the effects of bioreactive BZ metabolites on the myeloid lineage, Significantly, BQ induces an incomplete program of granulopoiesis in myeloblasts, whether in vivo or in culture [15], that results in differentiation of the myeloblasts only to the proliferating myelocyte stage. BQ has also been shown to inhibit recombinant CPP32 (Fig. 11), an essential enzyme in the apoptotic process, suggesting that BQ, in 32D cells and in vivo, prevents physiological cell death in a proliferating population of myelocytes. Taken together, this would result in a clone of myeloid progenitors that has uncontrolled expansion and lacks the capacity to differentiate to mature neutrophils and thus has characteristics of the acute leukemic phenotype.

Physiological induction of terminal granulocytic differentiation in myeloblasts by G-CSF results from the upregulation of leukotriene D₄ production, as essential intracellular mediator of G-CSF signaling. HQ via BQ binds to the leukotriene D₄ receptor [31], most probably in a covalent manner to constitutively activate this signal-driven process. Consequently, in the bone marrow, HQ, in the presence of leukotriene D₄, shifts the stage-specific pattern of terminal differentiation induced by leukotriene D₄ to the incomplete (myelocyte) profile induced by HQ [31]. The constitutive activation of this essential signaling pathway might contribute to BZ's ability to induce acute myeloid leukemia. In support of this speculation, it has been reported that constitutive overexpression of the c-kit receptor in 32D myeloblasts results in a population of cells that when injected into the bone marrow of syngeneic mice results in acute leukemia in four to six weeks [51]. Experiments to test the leukemogenic capacity of HQ-treated, incompletely differentiated 32D myeloblasts in syngeneic mice are in progress.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
We are greatly indebted to Dr. Srinivasa Srinivasula of Dr. Emad Alnemri's laboratory, Kimmel Cancer Institute, for carrying out the experiment which tested the effect of BQ on the protease activity of CPP32. We are indebted to Dr. Alan Friedman for the kind gift of the 32D cl 3(G) clone. We are also indebted to Drs. James Karaszkiewicz and Marlene Darfler for carrying out the in situ detection of apoptosis by DAPI staining.

Betsy A. Hazel was supported by a Grant-in-Aid from the American Petroleum Institute.

This paper was presented at the Eighty-Seventh Annual Meeting of the American Association for Cancer Research, Washington, DC, April 24, 1996, and was taken, in part, from a thesis presented by Christine Baum to the Fachbereich of Human Medizin of Phillips Universität, Marburg, Germany in partial fulfillment of the M.D. degree.

	Footnotes

 
Provisionally accepted July 5, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

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