Cancer Letters, 62 (1992) 225 - 232

Elsevier Scientific Publishers Ireland Ltd.

225

Crocidolite asbestos suppresses the differentiation of HL-60 cells induced by DMSO

Ayako Ueki”, Hirobumi Tsushimaa, Fuminori Hyodoh”, Keigo Kinugawa”, Masabumi

Tomitab, Junko Kazahayaa and Risa ShiratoC

“Department of Hygiene, bDepartment of Legal Medicine and ‘Department of Medicine, Kawasaki Medical School, Kurashiki (Japan)

(Received 16 August 1991)

(Revision received 22 November 1991) (Accepted 26 November 1991)

Summary Introduction

This study was undertaken to determine if the biological function of inducers for cell dif- ferentiation is affected by asbestos fibers, which are sometimes deposited in human tissues. Protein kinase C actiuity, c-myc protein ex- pression and cell surface CR3 expression were used as the markers of cell differentiation. The function of dimethylsulfoxide (DMSO), an in- ducer of cell differentiation, was suppressed by the co-culturing of crocidolite asbestos, because DMSO reacted with the hydroxyl radical released after the stimulation with crocidolite and spent itself. Superoxide dismutase (SOD) inhibited the effect of crocidolite, reacting rapidly with ’ 0 ; before the secondary release of ‘OH. Asbestos fibers deposited in tissues may inhibit the function of inducers which stimulate immature cells to dif- ferentiate, because such inducers frequently are also radical scavengers.

The release of radicals plays an important role in the cytotoxicity and tumor promotion caused by asbestos fibers [l]. Recent studies have shown that asbestos-induced cytotoxicity can be suppressed by the scavengers of superoxide or the hydroxyl radical [Z]. Some of these radical scavengers, e.g. DMSO and retinoic acid, also induce cell differentiation.

In this study, the authors sought to deter- mine if the biological function of inducers for cell differentiation could be suppressed by asbestos fibers, which are sometimes deposited in human tissues, as a stimulator of radical release.

Keywords: HL-60 cells; cell differentiation; protein kinase C; radicals; asbestos

Correspondence to: A. Ueki, Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki

701-01, Japan.

The human promyelocytic cell line HL-60 constitutes a useful system for the study of cell differentiation, since it can be induced to dif- ferentiate to mature myeloid cells with DMSO [3] or retinoic acid [4]. Increased protein kinase C (PKC) activity [5,6], and marked diminution of c-myc gene expression [7] dur- ing HL-60 cell differentiation induced by DMSO and/or retinoic acid have been reported. Several kinds of surface antigens, such as CRl, CR3 and FcR, become detec- table with the differentiation of HL-60 cells [8].

In this study, PKC activity, c-myc protein ex- pression, and cell surface CR3 expression

0304-3835/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd

Printed and Published in Ireland

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were used as the markers of cell differentiation. The induction of cell differentiation in HL-60 cells by DMSO was suppressed when asbestos (crocidolite) was added-to the culture system, but the effect disappeared following co- culturing with SOD.

Materials and Methods

Reagents _ Propidium iodide (PI), dimethylsulfoxide

(DMSG) , ethylenediamine-tetraacetic acid (EDTA), ethyleneglycol bis (Z-aminoethyl- ether) tetraacetic acid (EGTA) and phenyl- methanesulfonyl fluoride (PMSF) were pur- chased from Nacarai Tesque (Kyoto, Japan). Fluorescein diacetate (FDA), phorbol myristate acetate (PM&, dimethylpyrroline oxide (DMPO), NADH, Nonidet P-40, Tween-20, and bovine erythrocyte superoxide dismutase (SOD) were purchased from Sigma (St. Louis, MO). Monoclonal antibody (mAb) to C3R, and PE conjugated anti-mouse IgG antibody purchased from Becton Dickinson (Mountain View, CA), mAb to c-myc protein (Cambridge Research Biochemicals, Cambridge, U.K.), biotinylated goat anti-mouse IgG (Tago, Bur- lingame, CA), and FITC-avidin (Vector Lab. Burlingame, CA) were also used in the ex- periments. Crocidolite asbestos (UICC stand- ard reference sample) was kindly given to us by the Pneumoconiosis Research Unit, the Na- tional Center for Occupational Health, South Africa.

Cell culture HL-60 cells were maintained in RPM1 1640

supplemented with 15% fetal calf serum in a humidified atmosphere of 5% CO*. All cultures were initiated with 2 x lo5 cells/ml in plastic petri dishes. HL-60 cells were induc- ed to differentiate with 1.25% DMSO for 4- 10 days.

Incubation of HL-60 cells with crocidolite Crocidolite fiber was autoclaved and used at

a final concentration of 25, 50, 100 or 200

pg/ml with or without the addition of DMSO. After incubation, cells were harvested and washed with 10 mM EDTA-PBS (pH 7.4), to separate the attached cells from crocidolite fibers. In some cases, the culture was sup- plemented with SOD at a final concentration of 50 - 400 U/ml.

Electron paramagnetic resonance (EPR) spec- trometry

For the detection of ‘OS, ‘OH or ‘CHs, the electron paramagnetic resonance (EPR) spin- trapping technique was used by employing 5,5-dimethyl-1-pyrroline-l-oxide (DMPO) as a radical trapping reagent [9, lo]. HL-60 cells were induced to differentiate with 1.25% DMSO for 10 days, and suspended in Hank’s balanced salt solution. Then we transferred 150 ~1 of a mixture of the cell suspension (1 x lo7 cells/ml), stimulus (100 - 500 ng/ml PMA or 50 pg/ml crocidolite), and DMPO (1% , 0.09 M) to a flat quartz EPR cuvette. SOD was used at a final concentration of 100 units/ml. For detection of ‘CHs a mixture of the cell suspension, crocidolite, DMPO and DMSO (1.25%) was used for the experiments. Spectrometry was performed using an EPR spectrophotometer (JEOL. JES-REZX).

PKC assay HL-60 cells (1 x 107) were harvested,

washed with 10 mM EDTA-PBS, and disrupted by sonication in a homogenization buffer (PBS containing 10 mM EGTA, 20 mM EDTA, and 1 mM PMSF). The homogenates were centrifuged at 100 000 x g for 1 h and the supernatant was used as the source of cytosolic enzyme. The pellet was resuspended in a homogenization buffer containing 1% NP-40, incubated on ice for 30 min, and cen- trifuged at 100 000 x g for 1 h. The superna- tant was used as the source of membrane bound enzyme. The PKC assay was perform- ed using a PKC Assay System (Amersham, U.K.) and the protein concentration was measured with protein assay reagent (Pierce, Rockford, IL) _

227

Quantitafion of CR3 After induction of differentiation with

DMSO, cells were stained with anti-CR3 mAb and PE-labelled goat anti-mouse IgG antibody. The percentage of positive cells and .the fluorescence intensity were estimated using a FACStar model flow cytometer (Becton Dickinson, Mountain View, CA)

c-myc Protein expression HL-60 cells were fixed with cold ethanol

containing 1% acetic acid for 5 min, washed with 0.5% Tween 20-PBS, and stained with anti-c-myc protein mAb, biotinylated goat anti- mouse IgG, and FITC-avidin at 4OC. The percentage of positive cells was calculated by flow cytometry.

Viability analysis and total cell counting Cellular viability was determined by flow

cytometry using the method of Rotman and Papermaster [ll]. Cells were stained with 5 pg/ml of FDA and 50 pg/ml of PI, and analys- ed by flow cytometry as soon as possible. The counting of the absolute number of cells re- maining at the end of incubation was perform- ed using a hemocytometer.

Lactate dehydrogenase (LDH) assay The LDH assay was performed using the

LDH assay system (Yatoron, Tokyo, Japan), a modified method of Hill [12]. Cell culture supernatant was added to the mixture of 0.025 M pyruvate, 0.1 M sodium phosphate buffer (pH 7.2) and 1 mg/ml DPNH solution. Decrease in optical density at 340 nm was employed as a measure of enzyme activity.

Statistical analysis Data were analysed for statistical significance

by the Wilcoxon test. P < 0.05 was consid- ered significant.

Results

Total cell count and cell viability The total cell number increased during 2, 3,

and 5 days of incubation. A significant decline in the growth rate was observed in cells induc- ed to differentiate with DMSO, compared with control cells (P < 0.05) or cells incubated with DMSO plus crocidolite (P < 0.05, Table la). Neither incubation of ceils with 50 pg/ml of crocidolite nor induction of differentiation with 1.25% DMSO was cytotoxic (Table lb). No significant difference in LDH release was observed between cells incubated with DMSO alone and the cells incubated with both DMSO and crocidolite (Table lc).

EPR spectra The DMPO-OOH signal was detected after

the addition of PMA, and was significantly reduced in the presence of SOD or DMSO (Fig. 1A). After stimulation with crocidolite asbestos alone a DMPO-OH signal was observ- ed (Fig. lB), and it was reduced with DMSO, one of the ‘OH scavengers (Fig. 1C). The DMPO-OH signal was markedly decreased in the presence of SOD, a ‘0; scavenger (Fig. 1C). As shown in Fig. lE, two spin-trapped adducts (DMPO-OH and DMPO-CH3) were detected after incubation of DMSO with the crocidolite and DMPO mixture of cells; this fact suggests the conversion of DMSO to CH4 after the reaction with ‘OH.

PKC actiuity A significant increase in PKC activity was

detected in HL-60 cells 3 days after the addi- tion of 1.25% DMSO. Cytosolic enzyme activ- ity (per mg protein) increased more than three times in 3 days (P < O.Ol), and a parallel in- crease in activity in membrane extracts was also observed. The PKC activity in cells treated with both DMSO and crocidolite was situated between that of control cells and that of cells treated with DMSO alone. But PKC activity in- creased to almost the same level as in the DMSO group when SOD was added to the culture system simultaneously with DMSO and crocidolite (Table II).

CR3 expression The percentage of CR3 positive cells and

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Table 1. (a) Absolute number of cells (X 10 -5/ml).

DMSO cc Incubation (days)

2 3 5

- 43.7 f 3.1 56.5 ZIZ 9.7 120.9 f 10.7 l

+ - 36.1 zt 3.1 55.4 l 10.7 88.0 f 7. J

+ + 40.4 l 7.7 61.0 f 8.8 105.2 f 29.9 1 l

(b) % Cell viability. ‘*’

DMSO Concentration of crocidolite (pg/ml)

0 25 50 100 200

+ 96.0 f 0.6 94.9 f 0.3 94.4 l 0.3 92.2 + 0.6’ 90.8 + 0.7’ 95.9 l 1.0 95.2 f 0.4 94.2 l 0.5 91.8 f 0.7’ 91.5 f 1.1’

(c) LDH release from cells.“‘b

Incubation (days) LDH activity (U/lo6 cells)

DMSO ( + ) crocidolite ( - ) DMSO ( + ) crocidolite ( + )

2 69.5 zt 7.2 72.8 f 8.9 3 105.5 + 22.8 98.2 f 11.9 4 124.0 f 17.1 118.8 f 42.0

Cell free medium 47.0.

n = 6, mean f S.D.

“DMSO: 1.25%.

blncubation: 3 days. ‘Incubation: 4 days.

mean fluorescence intensity increased in dif- ferentiated cells incubated with DMSO (Table III). With the addition of crocidolite (25 or 50 pg/ml) to cells being induced to differentiate with 1.25% DMSO, the percentages of CR3 positive cells decreased significantly (P C

0.05).

percentage of c-myc protein positive cells was about 65% (P < 0.05) of total cells. When crocidolite (50 pg/ml) was added to the culture together with DMSO, c-myc protein positive cells increased to about 88% of total cells (Table IV).

c-myc Protein expression Discussion

More than 90% of HL-60 cells expressed c- myc protein. After the induction of differentia- tion with 1.25% DMSO for 4 days, the

According to Hansen and Mossman [l], the exposure of rat macrophages to asbestos causes a significant increase in superoxide

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Fig. 1. EPR spectra studied with HL-60 cells. Each scan took 2 min. Cells were previously induced to differentiate with 1.25% DMSO for 10 days. (A) EPR spectra with PMA: (1) Before addition of PMA, 10 min after addition of; (2) PMA (100 ng/ml); (3) PMA and SOD (100 U/ml); (4) PMA and DMSO (1.25%) to cell suspension (1 x 107/ml) supplemented with 1% DMPO. (B) EPR spectra with crocidolite: (1) Before stimulation; (2) 1 min; (3) 13 min; (4) 37

min after addition of crocidolite (50 pg/ml) to cell suspension (1 x 107/ml) supplemented with 1% DMPO. (C) The effect of radical scavengers on EPR spectra: (1) Before stimulation, 60 min after addition of; (2) crocidolite (50 pg/ml); (3) crocidolite and DMSO (1.25%); (4) crocidolite and SOD (100 units/ml) to cell suspension (1 x 107/ml) sup- plemented with 1% DMPO. (D) Cell free controls: Hank’s solution containing 1% DMPO (1); Hank’s solution containing

1% DMPO and 50 pg/ml crocidolite (2). (E) Detection of ‘CH, derived from DMSO: EPR spectrometry was performed using a mixture of cell suspension (1 x 107/ml), crocidolite (100 pg/ml), DMPO (1%) and DMSO (1.25%). Two spin-trapped adducts, - DMPO-OH and D DMPO-CHs were detected: (1) 1 min; (2) 7 min; (3) 18 min; (4) 36 min after incubation.

230

Table 2. PKC activity. Table 4. c-myc Protein positive cells.

DMSO cc c-myc protein cpm/mg protein

Control DMSO

dMS0 + cc

DMSO + cc + SOD

Cytosolic Membrane

Expl Exp2 Expl

11878 11430 5878 85176 35883 12446

63750 20039 6504

82600 36142 -

SOD: 50 U/ml, cc: crocidolite. Incubation: 3 days.

release from the cells. Recent studies show that asbestos-induced toxicity to tracheal epithelial cells, lung fibroblasts, and alveolar macro- phages in vitro can be inhibited by both superoxide dismutase, and the scavengers of the hydroxyl radical [13,14]. Mossman [15] also reported that synthetic analogs of vitamin A, a radical scavenger, inhibited the hyper-

Table 3. (a) CR3 after induction of differentiation.

DMSO L CR3 +

(46)

93.9 f 0.1 5.5 f 1.7 0.4 89.8 +z 5.9 5.1 f 1.2

0.8 96.8 zt 0.6 8.3 l 2.0 1.25 95.4 f 2.1 27.7 zt 4.7”

1.7 89.1 f 1.8’ 66.3 + 17.7”

(bj Effect of crocidolite on CR3.

Crocidolite

(as/ml)

CR3 positive cells (%)

Exp. 1 Exp. 2

0 22.5 zt 1.7 22.0 f 5.1 25 16.6 l 3.4’ 18.4 f 7.3

50 14.6 * 1.6’ 14.5 f 2.3’

l P c 0.05, n = 4, mean * S.D., DMSO: 1.25%. Incubation: 4 days. L: cell viability

- -

93.4 zt + - 65.3 zt 15.3 + + 88.4 f

1.6 (41;) 1 3” 7.6 1 l

Unstained cells 1.7 f 1.6

l *P < 0.01, ‘P < 0.05, n = 4, mean f S.D. Incubation: 4 days.

plasia and squamous metaplasia of tracheal epithelium.

By employing EPR spectrometry using a spin-trapping agent DMPO, the generation of ‘0; and ‘OH was evaluated during the respi- ratory burst of HL-60 cells induced to differen- tiate by DMSO. Generation of the hydroxyl radical has been detected during the stimula- tion of HL-60 cells with crocidolite. The ap- pearance of OH declines significantly after the addition of DMSO, which is a potent scavenger of ‘OH [16,17] and generates methane (CH,) after reaction with ‘OH [18]. In our experiments, generation of ‘Hs was detected after the reaction of DMSO with the mixture of HL-60 cells, crocidolite and DMPO. The generation of the hydroxyl radical is also inhibited by the addition of the ‘0; scavenger SOD, suggesting that ‘0; is the ini- tial product of the respiratory burst reaction, as stated by Ueno et al. [19].

According to Makowska et al. [5], the a, @ and y isozymes of PKC increase at least twofold in HL-60 cells exposed to DMSO for 48 h, and the increase is not limited to the cytosolic enzyme, but also detected in membrane-associated enzyme. The process of differentiation by DMSO is reported qualitatively similar to that in vivo [20]. In our experiments, cytosolic PKC activity increased more than three times after induction of dif- ferentiation, but not in the presence of crocidolite asbestos. This inhibiting effect of asbestos disappeared when SOD was added to the culture system. This could have been

due to the destruction of the radical- scavenging/differentiator DMSO by reaction with crocidolite-induced radicals.

Several kinds of surface antigens become detectable with differentiation in HL-60 cells [ 11,201. Although HL-60 cells carry little CR3, the differentiation to mature myeloid cells resulted in increased expression of CR3, which was a beneficial marker in this report. The in- duction of CR3 by DMSO was suppressed when the cells were co-cultured with crocidolite.

Prochownik and Kukowska [Zl] stated that down-regulation of c-myc gene expression plays an important role in the induction of cell differentiation. In our experiments, the expres- sion of c-myc protein decreased in accord with cell differentiation in HL-60 cells, but increased after the addition of crocidolite asbestos to the culture system.

Although the mechanisms of inhibition re- main to be clarified, it is speculated that the function of DMSO as a stimulator of cell dif- ferentiation declined, because it was used up to neutralize ‘OH stimulated by crocidolite. These effects of crocidolite were not detected after the addition of SOD, because SOD as a scavenger of ‘0, reacted rapidly before the secondary release of ‘OH, and DMSO remain- ed intact in the medium.

Acknowledgments

This study was supported by Kawasaki Medical School Grant No. l-204. The authors thank Miss Masayo Watanabe, Miss Hisae Towaki and Mrs Hiroko Watanabe for their ex- cellent technical assistance.

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