Induction of differentiation and apoptosis by dithizone in human myeloid leukemia cell lines

Leukemia Research 22 (1998) 405 – 412

Induction of differentiation and apoptosis by dithizone in human myeloid leukemia cell lines Junya Kohroki a, Norio Muto a, Tetsuya Tanaka a, Norio Itoh a, Akira Inada b, Keiichi Tanaka a,* a

Faculty of Pharmaceutical Sciences, Osaka Uni6ersity, 1 -6, Yamadaoka, Suita, Osaka 565, Japan b Faculty of Pharmaceutical Sciences, Setsunan Uni6ersity, Hirakata, Osaka 573 -01, Japan Received 28 July 1997; accepted 3 November 1997

Abstract We investigated the effect of diphenylthiocarbazone (dithizone) and its structurally related compounds on the differentiation and apoptosis of two human myeloid leukemia cell lines. Dithizone caused a time- and concentration-dependent induction of differentiation in both the promyelocytic leukemia cell line HL-60 cells and the myeloblastic leukemia cell line ML-1 cells, as measured by nitroblue tetrazolium (NBT) reducing activity. Morphological changes and esterase activities confirmed that this differentiation took place. The induction of differentiation required the addition of dithizone to the culture medium for at least 12 h. The differentiation inducing activity was inhibited by the preincubation of dithizone with various metal ions such as Pb2 + , Zn2 + , Cu2 + and Mn2 + ions, but not with Fe3 + and Mg2 + ions. In addition, the DNA extracted from dithizone-treated HL-60 cells showed a typical ladder pattern characteristic of apoptosis in agarose gel electrophoresis. A quantitative analysis of DNA fragmentation revealed that this apoptosis was concentration- and time-dependent in both the HL-60 and ML-1 cells. Dithizone-induced apoptosis was also inhibited by preincubation with Mn2 + ions, but not with Mg2 + ions. These results indicate that dithizone induces both differentiation and apoptosis in HL-60 and ML-1 cells through a unique mechanism including metal chelation. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Leukemia cell lines; Differentiation; Apoptosis; Chelator; Dithizone; Metal ions

1. Introduction Human myeloid leukemia cell line can be induced to differentiate into neutrophils or monocytes by treatment with various agents, such as dimethylsulfoxide [1], 12-O-tetradecanoylphorbol-13-acetate (TPA) [2], alltrans-retinoic acid (ATRA) [3], 1a,25-dihydroxyvitamin D3 [4] and tumor necrosis factor (TNF) [5]. Since differentiated cells lose their proliferative ability and immortality, differentiation inducers may be useful for the treatment of leukemia. In fact, ATRA was administered to patients with acute promyelocytic leukemia, and it induced complete remissions [6]. The above-mentioned cell lines are also useful models for the study of Abbre6iations: TPA, 12-O-tetradecanoylphorbol-13-acetate; ATRA, all-trans-retinoic acid; TNF, tumor necrosis factor; EDTA, ethylenediaminetetraacetic acid; NBT, nitroblue tetrazolium; FBS, fetal bovine serum. * Corresponding author. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 9 7 ) 0 0 1 9 0 - 2

leukemia cell differentiation and for the development of differentiation therapy for leukemia. The differentiation inducers ATRA and TPA have recently been shown to induce apoptosis in leukemia cells [7,8]. Apoptosis is a form of self-controlled cell death characterized by nucleosomal fragmentation and by several morphological changes which differ from those of necrosis [9]. Many tumors involve a deviation in the homeostatic control of the differentiation and apoptosis of cells. The induction of apoptosis in proliferative tumor cells may thus be useful as anticancer therapy. However, the relationship between differentiation and apoptosis is still unclear. Diphenylthiocarbazone (dithizone), a metal chelator, has been used as a reagent for chelation titration [10]. In our previous study [11], we found that dithizone can induce the differentiation of F9 cells, a mouse teratocarcinoma-derived embryonal carcinoma cell, as can other metal chelators including hinokitiol, tropolon and

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deferoxamine [12]. It was recently reported that ethylenediaminetetraacetic acid (EDTA), a well-known metal chelator, induced differentiation and suppressed the proliferation of HL-60 cells, and that these effects were inhibited by the addition of zinc ions [13]. In the present study, we found that dithizone induced both differentiation and apoptosis in the human promyelocytic leukemia cell line HL-60 and the human myeloblastic leukemia cell line ML-1, via its potent metal-chelating activity.

2.4. Assay of esterase acti6ity Both a-naphthyl acetate esterase activity and naphthol-AS-D-chloroacetate esterase activity were determined by the method of Yam et al. [15].

2.5. Obser6ation of morphological changes Morphological changes were microscopically observed in smears stained with Wright-Giemsa stain.

2.6. Quantification of DNA fragmentation 2. Materials and methods

2.1. Materials Dithizone, nitroblue tetrazolium (NBT), a-naphthyl acetate and naphthol-AS-D-chloroacetate were purchased from Nacalai Tesque (Kyoto, Japan). RPMI1640 medium was obtained from Nissui Pharmaceutical Co. (Tokyo, Japan) and fetal bovine serum (FBS) was from Filtron (Brooklyn, Australia). TPA was purchased from Funakoshi (Tokyo). Diphenylthiocarbazide, s-diphenylcarbazone and diphenylcarbazide were products of Tokyo Chemical Industry Co. (Tokyo). All metal salts were purchased from Nacalai Tesque and Wako Pure Chemical Industries (Osaka, Japan). Other compounds employed were of analytical grade from commercial sources.

2.2. Cell lines and cell culture HL-60 cells were obtained from Human Science Research Resources Bank (Osaka, Japan), and ML-1 cells were kindly provided by Hayashibara Biochemical Institute (Okayama, Japan). Cells were grown in RPMI1640 medium supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2-air.

2.3. Assay of NBT reducing acti6ity Cells (1×106 cells/60 mm dish) were cultured with various concentrations of dithizone in RPMI-1640 medium containing 10% FBS for 4 days, and then the cells’ NBT reducing activity was determined by the method of Sakashita et al. [14] with a slight modification. In brief, the cells were harvested by centrifugation and suspended in 100 ml of NBT solution (4 mg/ml). After the addition of 100 ml of TPA solution (2 mg/ml), the cell suspension was incubated at 37°C for 20 min, 200 ml of 1 N HCl was added at 4°C to terminate the reaction. After centrifugation, 600 ml of dimethylsulfoxide was added to the cell pellets to solubilize the formazan deposits. The amount of formazan formed was assayed spectrophotometrically at 560 nm in a spectrophotometer.

The extent of DNA fragmentation was determined by the method of Peradones et al. [16] with a slight modification. Briefly, cells were collected by centrifugation at 750× g for 5 min and lysed in 200 ml of lysis buffer (0.2% Triton X-100, 10 mM Tris–HCl (pH 7.5) and 1 mM EDTA) for 10 min on ice. The lysate was centrifuged at 13 000×g for 20 min to separate the supernatant containing fragmented DNA from the pellet containing intact DNA. Both the supernatant and the pellet, resuspended in 200 ml of lysis buffer, were mixed with 250 ml of isopropyl alcohol and 50 ml of 5 M NaCl, and then stored at − 30°C for 4 h to precipitate DNAs. After centrifugation at 12 000×g for 10 min, 500 ml of 4% perchloric acid was added to the DNA pellets, followed by incubation with 100 ml of 88 mM diphenylamine solution in glacial acetic acid, sulfuric acid and 1.6% acetaldehyde (v/v) at 90°C for 10 min. After storage in the dark at room temperature overnight, the absorbance of sample solution at 560 nm was measured in a spectrophotometer. The rate of DNA fragmentation was calculated as the percentage of fragmented DNA content to total DNA content.

2.7. Detection of DNA fragmentation by electrophoresis DNA ladder was detected according to the method of Peradones et al. [16] with slight modifications. At the time indicated, cells were collected by centrifugation at 750× g for 5 min and lysed in 400 ml of lysis buffer for 10 min on ice. The lysate was centrifuged at 13 000×g for 20 min to obtain the supernatant containing fragmented DNA. An aliquot (200 ml) of the supernatant and the pellet were used for the assay of DNA content. The other half was incubated with 1 mg/ml RNase A (Sigma, St. Louis, MO) for 60 min at 37°C, followed by treatment with 100 mg/ml proteinase K (Wako Pure Chemical Industries, Osaka, Japan) for an additional 45 min at 50°C. DNAs were preincubated by adding 50 ml of 5 M NaCl and 250 ml of isopropyl alcohol at − 30°C overnight. DNA samples were resolved by electrophoresis on a 2% agarose gel. After electrophoresis for 90 min at 50 V, the gel was stained with ethidium

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bromide, and the DNA was visualized by a UV transilluminator.

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Table 1 Effects of dithizone and its structurally related compounds on the induction of differentiation of HL-60 cells NBT reduction (A560/106cells)

3. Results

3.1. Induction of differentiation in human leukemia cell lines Dithizone and its structurally related compounds were examined for their differentiation-inducing activity in promyelocytic HL-60 cells and myeloblastic ML-1 cells at concentrations ranging from 0.16 to 100 mM. HL-60 cells were incubated with various concentrations of compounds for 4 days and assayed for their NBT reducing activity, which is a typical marker for the differentiation of myeloid leukemia cells [17]. Among the compounds tested, only dithizone produced high NBT reducing activity. As shown in Fig. 1A, HL-60 cells were induced to differentiate dose-dependently by

Fig. 1. Effects of dithizone on the induction of differentiation of leukemia cells. HL-60 cells (A) and ML-1 cells (B) were treated with various concentrations of dithizone for 4 days, and their NBT reducing activity levels were determined. Panel C: HL-60 cells were treated with 20 mM dithizone for the indicated times, followed by the determination of NBT reducing activity. Panel D: HL-60 cells were treated with 20 mM dithizone for the indicated times and cultured up to 4 days without dithizone. All data presented are means 9 S.D. of triplicate cultures.

Compound

None

20 mM

100 mM

Dithizone Diphenylthiocarbazide Diphenylcarbazone Diphenylcarbazide

0.042 9 0.006 0.067 9 0.007

0.187 90.018 0.056 9 0.008

– 0.063 9 0.014

0.047 9 0.013

0.056 9 0.012

0.062 9 0.014

0.046 90.010

0.032 90.004

0.032 9 0.004

HL-60 cells were treated with indicated concentrations of each compound for 4 days, and their NBT reducing activities were determined. Data presented are means 9S.D. of triplicate cultures.

dithizone at concentrations ranging from 10 to 20 mM. The three dithizone analogues, diphenylcarbazone, diphenylcarbazide and diphenylthiocarbazide, showed no effect even at the concentration of 100 mM (Table 1). A similar induction of differentiation by dithizone was demonstrated in the ML-1 cells at slightly higher concentrations, as shown in Fig. 1B. We investigated the time course of differentiation in HL-60 cells treated with 20 mM dithizone. As shown in Fig. 1C, NBT reducing activity was observed within 24 h after incubation with dithizone and reached a plateau between 48 and 96 h after the addition of dithizone. We next identified the onset time for the induction of differentiation by dithizone. HL-60 cells were cultured in the presence of dithizone for various times and shows then cultured for the remaining period in the absence of dithizone. Fig. 1D that irreversible differentiation was induced by treatment with dithizone for at least 12 h. Both of these cell lines were reported to be induced to differentiate into neutrophils by DMSO [1] and into monocytes by 1a,25-dihydroxyvitamin D3 [5]. Table 2 shows the esterase activities in HL-60 and ML-1 cells treated with 20 mM dithizone for 4 days. a-Naphthyl acetate esterase activity and naphthol-AS-D-chloroacetate esterase activity are typical markers for the differentiation of myeloid leukemia cells to monocytes and granulocytes, respectively [18]. In the present study, the percentage of a-naphthyl acetate esterase-positive cells in the HL-60 and ML-1 cells was increased to approximately 40% by the dithizone treatment, while that of the naphthol-AS-D-chloroacetate esterase-positive cells was weakly increased. Fig. 2 shows the morphological changes of HL-60 and ML-1 cells following exposure to 20 mM dithizone for 6 days. This treatment decreased the relative ratio of nuclei to cytoplasm in both cells, and especially it depressed the number of azurophilic granules in HL-60 cells. These results strongly indicated that dithizone induces HL-60 and ML-1 cells to undergo monocytic differentiation.

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Table 2 Effects of dithizone on the induction of esterase activities of HL-60 and ML-1 cells Positive cells (%) Cell lines

Compound

a-Naphthyl acetate esterase

Naphthol-AS-D-chloroacetate esterase

HL-60

None Dithizone None Dithizone

2.99 1.3 38.4 9 3.2* 3.9 9 1.3 38.39 5.4*

1.3 90.5 7.0 95.4 1.3 90.3 8.3 93.7**

ML-1

Cells were treated with 20 mM dithizone for 4 days, and the esterase activities of each cell line were determined. Data presented are means 9S.D. of triplicate cultures. * PB0.005 compared to non-treated cells. ** PB0.05 compared to non-treated cells.

Fig. 2. Morphological changes of HL-60 cells and ML-1 cells treated with dithizone. HL-60 cells (A and B) and ML-1 cells (C and D) were treated with (B and D) or without (A and C) 20 mM dithizone for 6 days. After treatment, cells were stained with Wright-Giemsa.

3.2. Inhibitory effect of metal ions on dithizone-induced differentiation To evaluate the contribution of the chelating ability of dithizone to the induction of differentiation, we examined the inhibitory effect of several metal ions. Each metal ion was preincubated at concentrations of 0.05–5 mM with 20 mM dithizone at 37°C for 30 min in the fresh culture medium. HL-60 cells were then exposed to this medium for 4 days, and their NBT reducing activity was assayed. As shown in Fig. 3, the dithizone-induced differentiation of HL-60 cells was effectively inhibited by preincubation with Mn2 + , Pb2 + , Zn2 + and Cu2 + ions, but not with Fe3 + or Mg2 + ions. Among these ions, Mn2 + appeared to be most potent, because it showed an inhibition even at 0.05 mM. The differentia-

tion-inducing ability of dithizone in ML-1 cells was similarly inhibited by preincubation with Mn2 + ions (Table 3).

3.3. Effects of dithizone on the induction of DNA fragmentation When dithizone was added to HL-60 cells at the concentration of 20 mM, the cell viability was reduced by approximately 50%, as determined by trypan blue dye exclusion test. Since most inducers of differentiation in leukemia cells are known to induce apoptosis [8,9], we examined whether the cell death caused by dithizone in HL-60 cells was due to apoptosis. We analyzed DNA fragmentation in dithizone-treated HL60 cells by agarose gel electrophoresis. Fig. 4 shows the

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Fig. 3. Effects of several metal ions on the dithizone-induced differentiation of HL-60 cells. 20 mM dithizone and 0.05 mM ( ), 0.5 mM (a) or 5 mM ( ) metal ions were preincubated for 30 min in the fresh medium. The cells were then cultured in those media for 4 days, and the cells’ NBT reducing activity was then determined. Data presented are means 9 S.D. of triplicate cultures. * Cytotoxicity.

appearance of DNA ladders in a dose-dependent manner. The extent of DNA fragmentation induced by dithizone was also quantified by a diphenylamine method. As shown in Fig. 5A, the extent of DNA fragmentation in the HL-60 cells was dose-dependently increased by dithizone, whereas that of the dithizone-treated ML-1 cells was weak (Fig. 5B). The dithizone-induced DNA fragmentation in the HL-60 cells occurred in a time-dependent manner (Fig. 5C), similarly to the profile of the time-dependent induction of differentiation by dithizone.

3.4. Inhibitory effect of Mn 2 + ions on dithizone-induced apoptosis of HL-60 We next examined the effect of the preincubation of dithizone with Mn2 + ions on the apoptosis in HL-60 cells. As shown in Fig. 6, the preincubation with 0.5 mM Mn2 + ions abolished the dithizone-induced DNA fragmentation in the HL-60 cells. In contrast to the Mn2 + ions, preincubation with Mg2 + ions had no effect on the dithizone-induced DNA fragmentation in HL-60 cells.

Table 3 Effect of Mn2+ ion on dithizone-induced differentiation of ML-1 cells Mn2+ (mM)

Relative activity (% of control)

0.05 0.5 5

78.2 9 10.3 30.7 9 4.5 18.7 9 1.7

20 mM dithizone and Mn2+ ion were preincubated for 30 min in the fresh medium. ML-1 cells were cultured in those media for 4 days, and the cells’ NBT reducing activity was then determined. Data presented are means 9S.D. of triplicate cultures.

Fig. 4. Detection of DNA fragmentation in HL-60 cells treated with dithizone by agarose gel electrophoresis. Lane 1, 100-bp ladder marker. Lane 2, control HL-60 cells. Cells were treated with 5 mM (lane 3), 10 mM (lane 4), 15 mM (lane 5), 20 mM (lane 6) or 25 mM (lane 7) dithizone for 4 days. After treatment, fragmented DNAs were analyzed by agarose gel electrophoresis.

4. Discussion In our previous study, we found that dithizone was able to induce both the differentiation and apoptosis of mouse embryonal carcinoma F9 cells, as did the other chelators hinokitiol and tropolon [12]. These activities of dithizone were effectively inhibited by preincubation with Fe3 + ion, indicating that iron chelation was essential in such cellular events. In the present study, we found a potent ability of dithizone to induce both the differentiation and apoptosis of two human myeloid leukemia cell lines. The induction of differentiation of HL-60 cells by dithizone was both time- and concentration-dependent. Esterase staining and the morphological features showed the differentiation of both cell lines to monocytes. Surprisingly, however, these inducing activities were not inhibited by preincubation with Fe3 + ion at the final concentration of 5 mM. This suggests that dithizone acts through a mechanism distinct from that observed in teratocarcinoma F9 cells [13]. In addition, dithizone did not induce the differentiation of KG-1 cells, another human myeloblastic leukemia cell line, as determined by NBT reducing activity and esterase activity (data not shown). In general, differentiation inducers have been reported to be effective for only some types of leukemia cells. For example, ATRA induced the differentiation of HL-60 and M1 cells, but not ML-1 or KG-1 cells [19]. TNF induced the differentiation of HL-60 [20], ML-1 [20]

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Fig. 5. Effects of dithizone on the induction of apoptosis of leukemia cells. HL-60 cells (A) and ML-1 cells (B) were treated with various concentrations of dithizone for 4 days, and their DNA fragmentation rates were then assessed. (C) HL-60 cells were treated with 20 mM dithizone for the indicated times, followed by the determination of the DNA fragmentation rate. All data presented are means 9 S.D. of triplicate cultures.

Fig. 6. Effects of metal ions on the dithizone-induced apoptosis of HL-60 cells. HL-60 cells were treated with 20 mM dithizone preincubated with 0.5 mM Mn2 + or 5 mM Mg2 + . After 4-day treatment, the DNA fragmentation rates of the cells were determined. Data presented are means9S.D. of triplicate cultures.

and M1 cells [21], but not K562 cells [20]. These findings suggest that such leukemia cells have distinct genetic or phenotypic cell potentials to differentiate. The details of this aspect of differentiation inducers remain to be investigated. In the present study, the cell proliferation and viability of HL-60 and ML-1 cells were markedly decreased by dithizone (data not shown). In earlier studies, the treatment of HL-60 cells with differentiation inducers was observed to induce apoptosis, together with differentiation [7,8]. We therefore examined the possibility that part of the cell death of HL-60 and ML-1 cells induced by dithizone was due to apoptosis. The detection of DNA ladders in the present agarose gel electrophoresis indicated that dithizone induced apoptosis in HL-60 cells. The quantitative analysis of DNA frag-

mentation induced by dithizone showed a dose- and time-dependency, resembling the profiles observed in the differentiation assay. ML-1 cells treated with dithizone also underwent concentration-dependent DNA fragmentation with a slightly weak response compared to that in the differentiation profile. During the normal maturation process of myeloid, apoptosis occurs as a physiological event [22]. It is well known that matured granulocytes and erythrocytes undergo spontaneous apoptosis at the end of their lifespan [22,23]. It has been suggested that apoptosis induced during the maturation of myeloid and erythroid modulates the cell numbers prior to the migration of mature cells from the bone marrow into the circulation or the peripheral [24]. The dithizone-induced differentiation of HL-60 and ML-1 cells, therefore, may reflect such a physiological phe-

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nomenon or a cell renewal system. It was suggested that the differentiation inducers ATRA, dimethylsulfoxide and TPA induced differentiation followed by apoptosis, when HL-60 cells were treated with these chemicals [7,8]. Judging from the similarities in the time-course and dose-response profiles of both cellular events produced by dithizone treatment, we speculate that certain cell populations which are induced to differentiate by dithizone may undergo apoptosis more readily. A number of studies have recently shown that iron deprivation can induce the inhibition of DNA synthesis, differentiation and/or apoptosis in several kinds of proliferating cells. It has been found that EDTA can induce differentiation and suppress cell proliferation in HL-60 cells [13], and that deferoxamine induces apoptosis in HL-60 cells [25], CCRF-CEM cells [26], and teratocarcinoma F9 cells [27]. These activities were effectively abrogated by the presaturation of the chelators with Zn2 + or Fe3 + ions. We demonstrated here that the preincubation of dithizone with Mn2 + , Pb2 + , Zn2 + and Cu2 + ions, but not Fe3 + and Mg2 + ions, efficiently inhibited dithizone-induced differentiation in HL-60 and ML-1 cells. This is consistent with the theory that dithizone chelates Pb2 + , Zn2 + and Cu2 + ions more efficiently than Fe3 + ions [28], and suggests that the deprivation of essential metals other than iron may also induce differentiation and/or apoptosis in HL-60 and ML-1 cells, though the mechanism remains to be clarified. In contrast, the inhibitory effect of Mn2 + ions was so potent that the preincubation of 20 mM dithizone with 0.5 mM Mn2 + ions at 37°C for 30 min completely inhibited differentiation. Moreover, dithizone-induced apoptosis was also inhibited by pretreatment with Mn2 + ions at the same concentration. These findings suggest a unique mechanism, such as a functional disorder of manganese-containing protein, underlying the dithizone-induced differentiation and apoptosis in leukemia cell lines. However, dithizone is known to be easily oxidized to dehydrodithizone in aqueous solution containing some metal ions [29], suggesting that the pretreatment of dithizone with Mn2 + ions causes structural changes. We have preliminarily observed that Mn2 + ions remarked accelerate the oxidation of dithizone, and that other metal ions do not (unpublished data). Consequently, the probability that dithizone acts by inducing cellular manganese deprivation can be ruled out. Apoptosis was recently reported to occur in many types of cancer cells exposed to various chemotherapeutic drugs and cytotoxic agents [30]. These compounds are known to be disrupters of mitochondrial function and/or inhibitors of cell proliferation. It may therefore be very important to elucidate what kind of chemical reactivities of dithizone interfere with such cellular functions. This may reveal common pathway for the triggering of the induction of differentiation and/or apoptosis in cancer cells by chemical

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agents. Unfortunately, it has been suggested that dithizone causes diabetes in laboratory animals [31]. This indicates that it is difficult to utilize dithizone for patients with leukemia as an anti-cancer drug. However, these experimental findings obtained by clarifying the mechanism of dithizone-induced differentiation and/or apoptosis may provide some information to develop a new approach to the differentiation therapy.

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