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Cytotoxicity and apoptosis induction of sodium fluoride in human promyelocytic leukemia (HL-60) cells
Environmental Toxicology and Pharmacology 11 (2002) 85 – 91 www.elsevier.com/locate/etap
Cytotoxicity and apoptosis induction of sodium fluoride in human promyelocytic leukemia (HL-60) cells Je-Seon Song a, Hee-Yeon Lee a, Eunyong Lee a, Hyun Jin Hwang b, Jeong Hee Kim a,* a
Department of Oral Biochemistry and Institute of Oral Biology, College of Dentistry, Kyung Hee Uni6ersity, 1 Hoeki-Dong, DongDaeMoon-Ku, Seoul 130 -701, South Korea b Department of Chemistry, College of Literature and Science, Kyung Hee Uni6ersity, 1 Hoeki-Dong, DongDaeMoon-Ku, Seoul 130 -701, South Korea Received 20 June 2001; received in revised form 26 September 2001; accepted 30 September 2001
Abstract The role of sodium fluoride (NaF) in cytotoxicity and induction of apoptosis was investigated by treating human promyelocytic leukemia (HL-60) cells with varying concentrations of NaF, from 0 to 250 ppm for different periods (0 – 72 h). At lower concentrations (0–50 ppm), no significant cytotoxicity was observed in response to NaF treatment. However, at higher concentrations (100–250 ppm), NaF reduced cell viability, and decreased DNA and protein biosynthesis capability in cultured HL-60 cells. The growth inhibitory and antiproliferative effects of NaF appear to be attributable to its induction of apoptotic cell death, as NaF induced morphological changes, internucleosomal DNA fragmentation, and increased the proportion of hypodiploid cells. NaF treatment also gradually decreased the expression of the anti-apoptotic protein Bcl-2, and increased activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase. These results provides important information towards understanding the mechanism by which NaF mediates cytotoxicity and apoptosis. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sodium fluoride; Cytotoxicity; Apoptosis; HL-60 cells
1. Introduction Fluoride compounds are naturally present in soil, water and food. These compounds have also been used for many years as additives in toothpaste, mouthwash, and drinking water in order to reduce the incidence of caries. Although fluorides are normally used in low concentrations, the possible cytotoxic effects of these compounds must be considered. Several research groups have reported cytotoxic effects of fluoride compounds, although the sensitivity to these effects can vary, depending on the cell type and the pH of the medium (Helgeland and Leirsker, 1976; Holland, 1980). Sodium fluoride (NaF) has been reported to inhibit the growth of human diploid cells in culture (Oguro et al., 1990). It has also been reported that NaF treatment induces chromosomal aberrations * Corresponding author. Tel.: +82-2-961-0915; fax: + 82-2-9601457. E-mail address: [email protected] (J.H. Kim).
and unscheduled DNA synthesis in human diploid fibroblasts (JHU-1 cells) (Tsutsui et al., 1984) and chromosomal aberrations in rat bone marrow cells (Tsutsui et al., 1984; Khalil, 1995). Furthermore, it has been demonstrated that NaF is mutagenic in L578Y mouse lymphoma cells (Caspary et al., 1987) and that NaF can transform Syrian hamster cells (Jones et al., 1988). Recently, fluoride compounds have been reported to be cytotoxic and clastogenic in human foreskin fibroblasts (Hayaashi and Tsutsui, 1993). These effects occurred in a cell cycle-dependent manner, as little cytotoxicity was observed in cells in the G1 phase, whereas significant increases in chromosomal aberrations were observed in cells in early and middle S phase (Hayaashi and Tsutsui, 1993). Jeng et al. (1998) reported that the inhibition of protein synthesis by NaF was toxic to oral mucosal fibroblasts in vitro. NaF cytotoxicity has also been associated with general decreases in DNA, RNA and protein biosynthesis (Slamenˇova´ et al., 1992). It was suggested that cytotoxicity
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of fluoride was associated with apoptosis in rat liver alveolar macrophages and osteosarcoma cells (Hirano and Ando, 1996, 1997) and the involvement of caspase3 activation in human leukemia cells (Anuradha et al., 2000) and mitogen-activated protein kinase p38 in epithelial cells (Thrane et al., 2001) are reported. However, the genotoxic effects of fluoride compounds are still controversial. It has been reported that there is no effect of fluoride compounds on sister chromatid exchange in Chinese hamster ovary (CHO) cells, bone marrow (CHBM) cells (Li et al., 1987a,b), or in cultured rat bone marrow cells (Khalil and Da’dara 1994), even at cytotoxic concentrations. No chromosomal aberrations were observed in human diploid fibroblast cells treated with relatively low concentration of NaF (5 –10 ppm) (Tsutsui et al., 1995), and no mutagenic effects were observed in human EUE cells at 10–150 ppm of NaF (Slamenˇ ova´ et al., 1992). In order to gain a better understanding of fluoride toxicity, we characterized the cytotoxicity and the pattern of cell death induced by NaF in human promyelocytic leukemia (HL60) cells. These cells provide a useful model system to characterize the cytotoxic and/or apoptosis-inducing effects of various agents (Suh et al., 1995). NaF induced cytotoxicity and apoptosis at concentrations of 0– 250 ppm in this study. We also investigated the mechanisms underlying the induction of apoptosis, with a particular focus on the expression of Bcl-2, the activation of caspase-3, and the resulting cleavage of poly(ADP-ribose) polymerase (PARP).
2. Materials and methods
2.1. Cell culture and cytotoxicity test Human promyelocytic leukemia (HL-60, ATCC CCL240) cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids and antibiotics in a humidified incubator with 5% CO2/95% air at 37 °C. A253 (human submaxillary gland epidermoid carcinoma, ATCC HTB-41), KB (human oral epidermoid carcinoma, ATCC CCL 17), MG63 (human osteosarcoma, ATCC CRL-1427), and HOS (human osteogenic sarcoma, ATCC CRL-1543) cells are cultured in appropriate media as recommended by the supplier. The trypan blue dye exclusion test was routinely used to assess cell viability. Exponentially growing HL-60 cells were seeded at 5× 104 cells per well in a 96-well plate and treated with the indicated concentrations of NaF or vehicle, as described in the figure legends. After various periods of exposure, the general viability of cultured cells was determined by assaying the reduction of 3-(4,5-dimethyl-thiazol-2-yl)-
2,5-diphenyltetrazolium bromide to formazan (Monks et al., 1991). Experiments were performed in triplicate.
2.2. Morphological obser6ations Cells used in this study were constantly observed under an inverted phase-contrast microscope (Olympus, Japan). Logarithmically growing HL-60 cells were seeded in a 24-well plate at a concentration of 2×106 cells per ml and treated with NaF as described. Photomicrographs were taken after various periods of NaF treatment, as described in the figure legends.
2.3. Analysis of DNA synthesis To assess the cell proliferation, tritiated thymidine incorporation assay was used as a measure of DNA synthesis. Exponentially growing cells were treated with either various concentrations of NaF or with phosphate buffered saline (PBS) for 24 h. During the final 4 h of this incubation, [methyl-3H] thymidine (1 mCi/ml) was added to the cell culture. The cells were then harvested by centrifugation, rinsed with cold PBS, 10% trichloroacetic acid (TCA) and treated with 0.1 M NaOH/2% Na2CO3 at 37 °C for 30 min. The level of [3H]-thymidine incorporation was measured by liquid scintillation counting (Beckman LS6500, USA).
2.4. Analysis of protein biosynthesis A radioactively-labeled amino acid incorporation assay (Kim et al., 2001) was used to measure the cellular level of protein biosynthesis. Cells were cultured in the presence of various concentrations of NaF for 24 h, and the final 3 h of this incubation [3H]-L-proline (1 mCi/ml) was added to the cell culture. Cells were harvested by centrifugation for 5 min at 300×g at 4 °C, rinsed with ice-cold PBS and precipitated with 10% TCA. The suspension was filtered through glass microfiber filter disks (Whatman GF/A) and then washed with 10% TCA solution followed by ethanol. The filter disk were then dried and radioactivity was counted by liquid scintillating counting.
2.5. Purification of DNA and electrophoresis DNA was purified as described previously (Kim et al., 1997). Cells were grown at a density of 2× 106 cells per ml and treated with various concentrations of NaF for 0–48 h, as described in the figure legends. The resulting purified DNA fragments were subjected to electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining. The results shown are representative of those obtained in three independent experiments.
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2.6. Flow cytometry analysis of apoptosis Cells were treated with various concentrations of NaF between 0 and 48 h, as indicated in the figure legends. Cells were harvested by centrifugation at 750×g for 5 min. Cell pellets were rinsed with PBS, mixed with 1:1 (v/v) mixture of PBS and 0.2 M Na2HPO4-0.1 M citric acid (pH 7.5) and fixed with cold ethanol at 4 °C for 1 h. Fixed cells were washed with PBS and resuspended in a staining solution containing 10 mg/ml propidium iodide and 100 mg/ml of DNasefree RNase. The cell suspensions were incubated at 37 °C for 1 h in the dark and analyzed on a fluorescence-activated cell sorter (FACScaliber) flow cytometer (Becton Dickinson, USA).
2.7. Western blot analysis After NaF treatment, cells were washed with PBS and lysed in a buffer containing 20 mM Tris– Cl, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM NaVO3, 100 mM NaF, 10% glycerol, 1 mM EGTA, 10 mM Na pyrophosphate and 1 mM phenylmethylsulfonylfluoride, pH 7.5. Cell lysates were cleared of insoluble material by centrifugation and the protein content in the cleared lysates was determined (Lowry et al., 1951). Samples containing equal amounts of protein (50 mg for Bcl-2, 30 mg for caspas-3 and PARP observation) were subjected to SDS-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) for 2 h at 80 mA. Blots were probed with mouse monoclonal anti-human anti-Bcl-2 (Oncogene), anti-caspase-3 (Transduction Laboratories, Lexington, KY) and rabbit monoclonal anti-human anti-PARP (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Immunoreactivity was detected using either an anti-mouse (Santa Cruz Biotechnology) or anti-rabbit (Amersham, Chicago, IL) peroxidase-conjugated secondary immunoglobulin G antibody followed by enhanced chemiluminescence (ECL, Amersham). Experiments are repeated at least three times.
Fig. 1. Antiproliferative effect of NaF in HL-60 cells. (A) Cells were incubated with 0, 50, 100, 150, 200 or 250 ppm of NaF for 24 h and cell survival was measured by the MTT assay. (B) Cells were incubated with 100 ( ) or 200 ppm ( ) of NaF for 24, 48 or 72 h and cell survival was measured by MTT assay. Control cells were incubated for the indicated amount of time except addition of NaF. Data are expressed as mean 9S.D., N = 3.
relative number of cells after 24, 48, or 72 h of exposure to various concentrations of NaF, we found that cell survival progressively decreased as the duration of NaF treatment increased (Fig. 1B). For the comparison of the cytotoxic effects of NaF on other cancer cells, the cell survival of A253, KB, MG-63, and HOS cells after NaF treatment were determined (Fig. 2). The IC50 values determined after 48 h of NaF incubation on these cells ranged from 87.5 to 177.1 ppm. The effects of NaF on cell survival were assessed by measuring the incorporation of [3H]thymidine into newly synthesized DNA. In parallel with the suppression of cell viability, [3H]thymidine incorporation progressively decreased with increasing concentrations of NaF (Fig. 3A). After 24 h of incubation with 100 and 150 ppm of NaF, incorporation of [3H]thymidine into HL-60 cells was suppressed to 87 and 16% of control levels, respectively. At a lower dose of NaF (50 ppm) a slight increase in [3H]thymidine incorporation was observed. In order to investigate the effect of NaF on protein synthesis, we measured the incorporation of [3H]proline into HL-60 cells (Fig. 3B). NaF treatment dramatically decreased levels of [3H]proline incorporated into HL-60 cells. Incorporation of [3H] proline
3. Results
3.1. Effect of NaF on cell sur6i6al, DNA and protein biosynthesis We first evaluated the effect of NaF treatment on HL-60 cells by using an MTT assay. As shown in Fig. 1A, NaF significantly decreased the rate of cell survival in a dose-dependent manner at a concentration range of 0 –250 ppm. The concentration required for 50% inhibition of growth (IC50) after 24 h of treatment was determined to be 95.3 ppm. When we compared the
Fig. 2. IC50 values of NaF in various cancer cell lines. Cells were incubated with 0, 50, 100, 150, 200 or 250 ppm of NaF for 24 h and cell survival was measured by the MTT assay. IC50 values were determined as the concentration required for 50% inhibition of growth. Data are expressed as mean 9S.D., N =3.
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3.3. Flow-cytometric analysis of NaF-induced cell death
Fig. 3. Inhibitory effect of NaF on DNA (A) and protein (B) biosynthesis in HL-60 cells. Cells were incubated with 0, 50, 100, 150, 200 or 250 ppm of NaF for 24 h. Incorporation of (A) [3H]thymidine and (B) [3H]proline were determined at each dose. Data are expressed as mean9 S.D., N =4, PB 0.01.
was suppressed to 73 and 23% of control levels, in cells that were treated with 100 and 150 ppm of NaF, respectively. A slight increase in [3H]proline incorporation was observed in cells that were treated with 50 ppm of NaF.
The induction of apoptosis in NaF-treated HL-60 cells was further analyzed by flow-cytometric determination of DNA content. A typical feature of apoptosis is loss of DNA. This occurs as degraded DNA diffuses out of fixed cells after endonuclease cleavage. DNA content histograms obtained from propidium iodidestained HL-60 cells revealed that the percentage of cells with reduced DNA content progressively increased from 5 to 72% in cells exposed to NaF for various times, from 7 to 48 h, as shown in Fig. 6. Cells were treated with increasing concentrations of NaF, from 0 to 200 ppm for 24 h and then harvested for flow-cytometric analysis. At lower concentrations of NaF (B10 ppm), no hypodiploid cells were detected. However, 38 and 84% of cells were hypodiploid after treatment with 100 and 200 ppm of NaF, respectively. In parallel with this increase in the number of cells with a hypodiploid DNA content, there was a concomitant decrease in the number of cells with diploid DNA content.
3.2. Induction of apoptosis by NaF To determine whether the growth inhibitory and antiproliferative effects of NaF are associated with programmed cell death or apoptosis, we initially examined the morphological changes induced by NaF treatment in HL-60 cells. Microscopic analysis of cells treated with NaF indicated that they had undergone gross morphological changes. After 24 h of exposure to NaF at 200 ppm, typical apoptotic changes were observed, including cell shrinkage, chromatin condensation, and formation of apoptotic bodies (Fig. 4). Apoptotic cells were detected at 7 h after treatment and the relative number of apoptotic cells progressively increased with increasing exposure time (data not shown). The most extensively studied biochemical event that occurs in apoptotic cells is the cleavage of nuclear DNA. Double-stranded DNA cleavage occurs at the linker regions between nucleosomes to produce fragments that are multiples of 180– 200 bp (Wyllie, 1980; Umansky, 1982; Arends et al., 1990; Kim et al., 1997). As illustrated in Fig. 4, analysis of DNA extracted from NaF-treated HL-60 cells revealed a progressive time-dependent increase in the non-random fragmentation of DNA into a ladder of multiple internucleosomal fragment of approximately 180– 200 bp (Fig. 5A). Similarly, DNA fragmentation was increased in a dose-dependent manner. At lower concentrations of NaF, 1 or 10 ppm, little fragmentation of DNA was observed, whereas extensive DNA fragmentation occurred when cells were treated with 100 or 200 ppm of NaF (Fig. 5B).
Fig. 4. Light microscope photographs of NaF-treated HL-60 cells. Cells were treated with (A) with PBS or (B) with 200 ppm of NaF for 24 h. Photomicrographs show representative morphological changes that were observed under a phase-contrast microscope.
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fragment and a concomitant disappearance of the fullsized 116 kDa molecule (Fig. 7). Taken together, these findings suggest that NaF induces apoptosis through the down-regulation of Bcl-2 and activation of caspase-3.
4. Discussion
Fig. 5. Induction of fragmentation of nuclear DNA by NaF treatment. (A) HL-60 cells were treated with various concentrations of NaF, as indicated, for 48 h. (B) HL-60 cells were treated with 200 ppm of NaF for various times, as indicated. DNA was isolated from each sample and isolated, electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining.
3.4. Effect of NaF treatment on Bcl-2 expression, caspase-3 acti6ation and PARP clea6age In order to investigate the mechanism by which NaF causes apoptosis, we first evaluated whether Bcl-2 levels were regulated. Bcl-2 is an important regulator of apoptotic pathways (Reed, 1998). Western blot analysis revealed that exposure to 200 ppm of NaF slightly reduced Bcl-2 immunoreactivity levels in HL-60 cells, as shown in Fig. 7. We next examined whether the caspase-3 protease was involved in the NaF-induced cell death response. NaF treatment induced the proteolytic processing of caspase-3, thus we observed decrease in 32 kDa caspase-3 precursor in a time-dependent manner. Activation of caspase-3 leads to the cleavage a number of proteins, including poly (ADP-ribose) polymerase (PARP). Although PARP is not essential for cell death, the cleavage of PARP is another hallmark of apoptosis. Treatment of HL-60 cells with NaF (200 ppm) caused a time-dependent proteolytic cleavage of PARP, with a progressive accumulation of the 85 kDa
To varying extents, human beings and animals are continually exposed to fluoride compounds. In addition, the increased use of fluorides in industry, medicine and dentistry exposes human beings to even greater levels of fluoride compounds. Although there are some conflicting reports in the literature, it generally seems that fluoride compounds can induce chromosomal aberrations and genetic mutations in cultured mammalian cells (Tsutsui et al., 1984; Caspary et al., 1987; Jones et al., 1988; Hayaashi and Tsutsui, 1993; Khalil, 1995). Therefore, we attempted to analyze the cytotoxicity and the mechanism of cell death induced by NaF at relatively high doses in human promyelocytic leukemia HL-60 cells. The HL-60 cell line is one of the most widely used human cell lines for studying the cellular and molecular events involved in cell death, proliferation and differentiation (Collins, 1987; Kawase et al., 1996). In present study, little cytoxicity of NaF was observed at lower concentrations, in terms of cell viability, DNA and protein biosynthesis, nucleosomal DNA fragmentation and appearance of hypodiploid cells. In fact, we observed a slight increase in both DNA and protein biosynthesis at concentrations of NaFB50 ppm. It has been reported that exposure to NaF at concentrations ranging from 10 − 6 to 10 − 3 M increases DNA and protein biosynthesis in bone forming cells (Farley et al., 1983) and in rat osteoblasts in culture (Li and DenBestin, 1993). Although these concentrations are lower than those used in this study, it seems that at relatively low levels, NaF has a stimulatory effect on DNA and protein biosynthesis. Our present investigation clearly demonstrates that NaF treatment at 100–200 ppm inhibits cell proliferation and induces apoptosis in human promyelocytic leukemia HL-60 cells. Alterations in cell morphology, fragmentation of nucleosomal DNA, and the appearance of hypodiploid cells all indicate that NaF induces apoptosis in these cells. Apoptosis is a tightly regulated process, which involves changes in the expression of distinct genes. Association of fluoride cytotoxicity and apoptosis induction was suggested (Hirano and Ando, 1996, 1997). Our results of activation of caspase-3 in fluoride-treated cells are in accord with the previous report (Anuradha et al., 2000). It seems that the activation of caspase-3 results from the decrease in Bcl-2 level in fluoride treated cells. The proto-oncogene bcl-2 en-
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Fig. 6. Flow-cytometry analysis of NaF-treated HL-60 cells. (A) Cells were treated with various concentrations of NaF, as indicated, for 24 h. (B) Cells were treated with 200 ppm of NaF for the indicated periods of time. The DNA content of cells in each sample was determined by flow cytometric analysis.
codes a 26 kDa mitochondrial-associated protein that is one of the major genes involved in regulation of apoptosis. The bcl-2 gene product is an intracellular suppressor of apoptosis and thus serves a cyto-protective function in cells (Hengartner and Horvitz, 1994). Furthermore, bcl-2 -transfected cells are often rescued from apoptotic death (Hockenbery, 1995). Our results indicate that NaF treatment (up to 72 h) decreases Bcl-2 immunoreactivity levels in HL-60 cells. The induction of apoptosis by NaF may be caused by this reduction in Bcl-2 levels. In addition, the cleavage of caspase-3 appears to correlate with NaF induced apoptosis in HL-60 cells. Caspase-3 is a cysteine protease that exists as an inactive zymogen in cells. Caspase-3 becomes
activated by sequential proteolytic events that cleave the 32 kDa precursor at aspartic acid residues to generate an active heterodimer comprised of 20 and 12 kDa subunits (Nicholson et al., 1995). The activity of caspase-3 was confirmed by the cleavage of PARP. In summary, NaF, one of the most widely used fluoride compounds, revealed no significant cytotoxicity at lower doses. However, at relatively high doses, NaF induced apoptosis in HL-60 cells. Our data suggest that apoptosis was induced in these cells by a decrease in Bcl-2 immunoreactivity levels and a concomitant activation of caspase-3. Taken together, these findings provide important new insights into the possible molecular mechanisms by which NaF mediates cytotoxicity and apoptosis.
Acknowledgements This study has been carried out under the University Research Institute grant (98-005-F00208) from Korea Research Foundation. J.-S. Song is a graduate fellow and E. Lee is a post-doctoral fellow of Brain Korea 21 program of Ministry of Education, Korea. Fig. 7. Regulation of Bcl-2, caspase-3, and PARP immunoreactivity levels by NaF. HL-60 cells were treated with 200 ppm of NaF for the indicated periods of time. Cells were lysed, and samples containing equal amounts of protein were subjected to immunoblotting with anti-Bcl-2, anti-caspase-3, or anti-PARP antibodies, as described in Section 2.
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Cytotoxicity and apoptosis induction of sodium fluoride in human promyelocytic leukemia (HL-60) cells Je-Seon Song a, Hee-Yeon Lee a, Eunyong Lee a, Hyun Jin Hwang b, Jeong Hee Kim a,* a
Department of Oral Biochemistry and Institute of Oral Biology, College of Dentistry, Kyung Hee Uni6ersity, 1 Hoeki-Dong, DongDaeMoon-Ku, Seoul 130 -701, South Korea b Department of Chemistry, College of Literature and Science, Kyung Hee Uni6ersity, 1 Hoeki-Dong, DongDaeMoon-Ku, Seoul 130 -701, South Korea Received 20 June 2001; received in revised form 26 September 2001; accepted 30 September 2001
Abstract The role of sodium fluoride (NaF) in cytotoxicity and induction of apoptosis was investigated by treating human promyelocytic leukemia (HL-60) cells with varying concentrations of NaF, from 0 to 250 ppm for different periods (0 – 72 h). At lower concentrations (0–50 ppm), no significant cytotoxicity was observed in response to NaF treatment. However, at higher concentrations (100–250 ppm), NaF reduced cell viability, and decreased DNA and protein biosynthesis capability in cultured HL-60 cells. The growth inhibitory and antiproliferative effects of NaF appear to be attributable to its induction of apoptotic cell death, as NaF induced morphological changes, internucleosomal DNA fragmentation, and increased the proportion of hypodiploid cells. NaF treatment also gradually decreased the expression of the anti-apoptotic protein Bcl-2, and increased activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase. These results provides important information towards understanding the mechanism by which NaF mediates cytotoxicity and apoptosis. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sodium fluoride; Cytotoxicity; Apoptosis; HL-60 cells
1. Introduction Fluoride compounds are naturally present in soil, water and food. These compounds have also been used for many years as additives in toothpaste, mouthwash, and drinking water in order to reduce the incidence of caries. Although fluorides are normally used in low concentrations, the possible cytotoxic effects of these compounds must be considered. Several research groups have reported cytotoxic effects of fluoride compounds, although the sensitivity to these effects can vary, depending on the cell type and the pH of the medium (Helgeland and Leirsker, 1976; Holland, 1980). Sodium fluoride (NaF) has been reported to inhibit the growth of human diploid cells in culture (Oguro et al., 1990). It has also been reported that NaF treatment induces chromosomal aberrations * Corresponding author. Tel.: +82-2-961-0915; fax: + 82-2-9601457. E-mail address: [email protected] (J.H. Kim).
and unscheduled DNA synthesis in human diploid fibroblasts (JHU-1 cells) (Tsutsui et al., 1984) and chromosomal aberrations in rat bone marrow cells (Tsutsui et al., 1984; Khalil, 1995). Furthermore, it has been demonstrated that NaF is mutagenic in L578Y mouse lymphoma cells (Caspary et al., 1987) and that NaF can transform Syrian hamster cells (Jones et al., 1988). Recently, fluoride compounds have been reported to be cytotoxic and clastogenic in human foreskin fibroblasts (Hayaashi and Tsutsui, 1993). These effects occurred in a cell cycle-dependent manner, as little cytotoxicity was observed in cells in the G1 phase, whereas significant increases in chromosomal aberrations were observed in cells in early and middle S phase (Hayaashi and Tsutsui, 1993). Jeng et al. (1998) reported that the inhibition of protein synthesis by NaF was toxic to oral mucosal fibroblasts in vitro. NaF cytotoxicity has also been associated with general decreases in DNA, RNA and protein biosynthesis (Slamenˇova´ et al., 1992). It was suggested that cytotoxicity
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of fluoride was associated with apoptosis in rat liver alveolar macrophages and osteosarcoma cells (Hirano and Ando, 1996, 1997) and the involvement of caspase3 activation in human leukemia cells (Anuradha et al., 2000) and mitogen-activated protein kinase p38 in epithelial cells (Thrane et al., 2001) are reported. However, the genotoxic effects of fluoride compounds are still controversial. It has been reported that there is no effect of fluoride compounds on sister chromatid exchange in Chinese hamster ovary (CHO) cells, bone marrow (CHBM) cells (Li et al., 1987a,b), or in cultured rat bone marrow cells (Khalil and Da’dara 1994), even at cytotoxic concentrations. No chromosomal aberrations were observed in human diploid fibroblast cells treated with relatively low concentration of NaF (5 –10 ppm) (Tsutsui et al., 1995), and no mutagenic effects were observed in human EUE cells at 10–150 ppm of NaF (Slamenˇ ova´ et al., 1992). In order to gain a better understanding of fluoride toxicity, we characterized the cytotoxicity and the pattern of cell death induced by NaF in human promyelocytic leukemia (HL60) cells. These cells provide a useful model system to characterize the cytotoxic and/or apoptosis-inducing effects of various agents (Suh et al., 1995). NaF induced cytotoxicity and apoptosis at concentrations of 0– 250 ppm in this study. We also investigated the mechanisms underlying the induction of apoptosis, with a particular focus on the expression of Bcl-2, the activation of caspase-3, and the resulting cleavage of poly(ADP-ribose) polymerase (PARP).
2. Materials and methods
2.1. Cell culture and cytotoxicity test Human promyelocytic leukemia (HL-60, ATCC CCL240) cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids and antibiotics in a humidified incubator with 5% CO2/95% air at 37 °C. A253 (human submaxillary gland epidermoid carcinoma, ATCC HTB-41), KB (human oral epidermoid carcinoma, ATCC CCL 17), MG63 (human osteosarcoma, ATCC CRL-1427), and HOS (human osteogenic sarcoma, ATCC CRL-1543) cells are cultured in appropriate media as recommended by the supplier. The trypan blue dye exclusion test was routinely used to assess cell viability. Exponentially growing HL-60 cells were seeded at 5× 104 cells per well in a 96-well plate and treated with the indicated concentrations of NaF or vehicle, as described in the figure legends. After various periods of exposure, the general viability of cultured cells was determined by assaying the reduction of 3-(4,5-dimethyl-thiazol-2-yl)-
2,5-diphenyltetrazolium bromide to formazan (Monks et al., 1991). Experiments were performed in triplicate.
2.2. Morphological obser6ations Cells used in this study were constantly observed under an inverted phase-contrast microscope (Olympus, Japan). Logarithmically growing HL-60 cells were seeded in a 24-well plate at a concentration of 2×106 cells per ml and treated with NaF as described. Photomicrographs were taken after various periods of NaF treatment, as described in the figure legends.
2.3. Analysis of DNA synthesis To assess the cell proliferation, tritiated thymidine incorporation assay was used as a measure of DNA synthesis. Exponentially growing cells were treated with either various concentrations of NaF or with phosphate buffered saline (PBS) for 24 h. During the final 4 h of this incubation, [methyl-3H] thymidine (1 mCi/ml) was added to the cell culture. The cells were then harvested by centrifugation, rinsed with cold PBS, 10% trichloroacetic acid (TCA) and treated with 0.1 M NaOH/2% Na2CO3 at 37 °C for 30 min. The level of [3H]-thymidine incorporation was measured by liquid scintillation counting (Beckman LS6500, USA).
2.4. Analysis of protein biosynthesis A radioactively-labeled amino acid incorporation assay (Kim et al., 2001) was used to measure the cellular level of protein biosynthesis. Cells were cultured in the presence of various concentrations of NaF for 24 h, and the final 3 h of this incubation [3H]-L-proline (1 mCi/ml) was added to the cell culture. Cells were harvested by centrifugation for 5 min at 300×g at 4 °C, rinsed with ice-cold PBS and precipitated with 10% TCA. The suspension was filtered through glass microfiber filter disks (Whatman GF/A) and then washed with 10% TCA solution followed by ethanol. The filter disk were then dried and radioactivity was counted by liquid scintillating counting.
2.5. Purification of DNA and electrophoresis DNA was purified as described previously (Kim et al., 1997). Cells were grown at a density of 2× 106 cells per ml and treated with various concentrations of NaF for 0–48 h, as described in the figure legends. The resulting purified DNA fragments were subjected to electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining. The results shown are representative of those obtained in three independent experiments.
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2.6. Flow cytometry analysis of apoptosis Cells were treated with various concentrations of NaF between 0 and 48 h, as indicated in the figure legends. Cells were harvested by centrifugation at 750×g for 5 min. Cell pellets were rinsed with PBS, mixed with 1:1 (v/v) mixture of PBS and 0.2 M Na2HPO4-0.1 M citric acid (pH 7.5) and fixed with cold ethanol at 4 °C for 1 h. Fixed cells were washed with PBS and resuspended in a staining solution containing 10 mg/ml propidium iodide and 100 mg/ml of DNasefree RNase. The cell suspensions were incubated at 37 °C for 1 h in the dark and analyzed on a fluorescence-activated cell sorter (FACScaliber) flow cytometer (Becton Dickinson, USA).
2.7. Western blot analysis After NaF treatment, cells were washed with PBS and lysed in a buffer containing 20 mM Tris– Cl, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM NaVO3, 100 mM NaF, 10% glycerol, 1 mM EGTA, 10 mM Na pyrophosphate and 1 mM phenylmethylsulfonylfluoride, pH 7.5. Cell lysates were cleared of insoluble material by centrifugation and the protein content in the cleared lysates was determined (Lowry et al., 1951). Samples containing equal amounts of protein (50 mg for Bcl-2, 30 mg for caspas-3 and PARP observation) were subjected to SDS-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) for 2 h at 80 mA. Blots were probed with mouse monoclonal anti-human anti-Bcl-2 (Oncogene), anti-caspase-3 (Transduction Laboratories, Lexington, KY) and rabbit monoclonal anti-human anti-PARP (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Immunoreactivity was detected using either an anti-mouse (Santa Cruz Biotechnology) or anti-rabbit (Amersham, Chicago, IL) peroxidase-conjugated secondary immunoglobulin G antibody followed by enhanced chemiluminescence (ECL, Amersham). Experiments are repeated at least three times.
Fig. 1. Antiproliferative effect of NaF in HL-60 cells. (A) Cells were incubated with 0, 50, 100, 150, 200 or 250 ppm of NaF for 24 h and cell survival was measured by the MTT assay. (B) Cells were incubated with 100 ( ) or 200 ppm ( ) of NaF for 24, 48 or 72 h and cell survival was measured by MTT assay. Control cells were incubated for the indicated amount of time except addition of NaF. Data are expressed as mean 9S.D., N = 3.
relative number of cells after 24, 48, or 72 h of exposure to various concentrations of NaF, we found that cell survival progressively decreased as the duration of NaF treatment increased (Fig. 1B). For the comparison of the cytotoxic effects of NaF on other cancer cells, the cell survival of A253, KB, MG-63, and HOS cells after NaF treatment were determined (Fig. 2). The IC50 values determined after 48 h of NaF incubation on these cells ranged from 87.5 to 177.1 ppm. The effects of NaF on cell survival were assessed by measuring the incorporation of [3H]thymidine into newly synthesized DNA. In parallel with the suppression of cell viability, [3H]thymidine incorporation progressively decreased with increasing concentrations of NaF (Fig. 3A). After 24 h of incubation with 100 and 150 ppm of NaF, incorporation of [3H]thymidine into HL-60 cells was suppressed to 87 and 16% of control levels, respectively. At a lower dose of NaF (50 ppm) a slight increase in [3H]thymidine incorporation was observed. In order to investigate the effect of NaF on protein synthesis, we measured the incorporation of [3H]proline into HL-60 cells (Fig. 3B). NaF treatment dramatically decreased levels of [3H]proline incorporated into HL-60 cells. Incorporation of [3H] proline
3. Results
3.1. Effect of NaF on cell sur6i6al, DNA and protein biosynthesis We first evaluated the effect of NaF treatment on HL-60 cells by using an MTT assay. As shown in Fig. 1A, NaF significantly decreased the rate of cell survival in a dose-dependent manner at a concentration range of 0 –250 ppm. The concentration required for 50% inhibition of growth (IC50) after 24 h of treatment was determined to be 95.3 ppm. When we compared the
Fig. 2. IC50 values of NaF in various cancer cell lines. Cells were incubated with 0, 50, 100, 150, 200 or 250 ppm of NaF for 24 h and cell survival was measured by the MTT assay. IC50 values were determined as the concentration required for 50% inhibition of growth. Data are expressed as mean 9S.D., N =3.
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3.3. Flow-cytometric analysis of NaF-induced cell death
Fig. 3. Inhibitory effect of NaF on DNA (A) and protein (B) biosynthesis in HL-60 cells. Cells were incubated with 0, 50, 100, 150, 200 or 250 ppm of NaF for 24 h. Incorporation of (A) [3H]thymidine and (B) [3H]proline were determined at each dose. Data are expressed as mean9 S.D., N =4, PB 0.01.
was suppressed to 73 and 23% of control levels, in cells that were treated with 100 and 150 ppm of NaF, respectively. A slight increase in [3H]proline incorporation was observed in cells that were treated with 50 ppm of NaF.
The induction of apoptosis in NaF-treated HL-60 cells was further analyzed by flow-cytometric determination of DNA content. A typical feature of apoptosis is loss of DNA. This occurs as degraded DNA diffuses out of fixed cells after endonuclease cleavage. DNA content histograms obtained from propidium iodidestained HL-60 cells revealed that the percentage of cells with reduced DNA content progressively increased from 5 to 72% in cells exposed to NaF for various times, from 7 to 48 h, as shown in Fig. 6. Cells were treated with increasing concentrations of NaF, from 0 to 200 ppm for 24 h and then harvested for flow-cytometric analysis. At lower concentrations of NaF (B10 ppm), no hypodiploid cells were detected. However, 38 and 84% of cells were hypodiploid after treatment with 100 and 200 ppm of NaF, respectively. In parallel with this increase in the number of cells with a hypodiploid DNA content, there was a concomitant decrease in the number of cells with diploid DNA content.
3.2. Induction of apoptosis by NaF To determine whether the growth inhibitory and antiproliferative effects of NaF are associated with programmed cell death or apoptosis, we initially examined the morphological changes induced by NaF treatment in HL-60 cells. Microscopic analysis of cells treated with NaF indicated that they had undergone gross morphological changes. After 24 h of exposure to NaF at 200 ppm, typical apoptotic changes were observed, including cell shrinkage, chromatin condensation, and formation of apoptotic bodies (Fig. 4). Apoptotic cells were detected at 7 h after treatment and the relative number of apoptotic cells progressively increased with increasing exposure time (data not shown). The most extensively studied biochemical event that occurs in apoptotic cells is the cleavage of nuclear DNA. Double-stranded DNA cleavage occurs at the linker regions between nucleosomes to produce fragments that are multiples of 180– 200 bp (Wyllie, 1980; Umansky, 1982; Arends et al., 1990; Kim et al., 1997). As illustrated in Fig. 4, analysis of DNA extracted from NaF-treated HL-60 cells revealed a progressive time-dependent increase in the non-random fragmentation of DNA into a ladder of multiple internucleosomal fragment of approximately 180– 200 bp (Fig. 5A). Similarly, DNA fragmentation was increased in a dose-dependent manner. At lower concentrations of NaF, 1 or 10 ppm, little fragmentation of DNA was observed, whereas extensive DNA fragmentation occurred when cells were treated with 100 or 200 ppm of NaF (Fig. 5B).
Fig. 4. Light microscope photographs of NaF-treated HL-60 cells. Cells were treated with (A) with PBS or (B) with 200 ppm of NaF for 24 h. Photomicrographs show representative morphological changes that were observed under a phase-contrast microscope.
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fragment and a concomitant disappearance of the fullsized 116 kDa molecule (Fig. 7). Taken together, these findings suggest that NaF induces apoptosis through the down-regulation of Bcl-2 and activation of caspase-3.
4. Discussion
Fig. 5. Induction of fragmentation of nuclear DNA by NaF treatment. (A) HL-60 cells were treated with various concentrations of NaF, as indicated, for 48 h. (B) HL-60 cells were treated with 200 ppm of NaF for various times, as indicated. DNA was isolated from each sample and isolated, electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining.
3.4. Effect of NaF treatment on Bcl-2 expression, caspase-3 acti6ation and PARP clea6age In order to investigate the mechanism by which NaF causes apoptosis, we first evaluated whether Bcl-2 levels were regulated. Bcl-2 is an important regulator of apoptotic pathways (Reed, 1998). Western blot analysis revealed that exposure to 200 ppm of NaF slightly reduced Bcl-2 immunoreactivity levels in HL-60 cells, as shown in Fig. 7. We next examined whether the caspase-3 protease was involved in the NaF-induced cell death response. NaF treatment induced the proteolytic processing of caspase-3, thus we observed decrease in 32 kDa caspase-3 precursor in a time-dependent manner. Activation of caspase-3 leads to the cleavage a number of proteins, including poly (ADP-ribose) polymerase (PARP). Although PARP is not essential for cell death, the cleavage of PARP is another hallmark of apoptosis. Treatment of HL-60 cells with NaF (200 ppm) caused a time-dependent proteolytic cleavage of PARP, with a progressive accumulation of the 85 kDa
To varying extents, human beings and animals are continually exposed to fluoride compounds. In addition, the increased use of fluorides in industry, medicine and dentistry exposes human beings to even greater levels of fluoride compounds. Although there are some conflicting reports in the literature, it generally seems that fluoride compounds can induce chromosomal aberrations and genetic mutations in cultured mammalian cells (Tsutsui et al., 1984; Caspary et al., 1987; Jones et al., 1988; Hayaashi and Tsutsui, 1993; Khalil, 1995). Therefore, we attempted to analyze the cytotoxicity and the mechanism of cell death induced by NaF at relatively high doses in human promyelocytic leukemia HL-60 cells. The HL-60 cell line is one of the most widely used human cell lines for studying the cellular and molecular events involved in cell death, proliferation and differentiation (Collins, 1987; Kawase et al., 1996). In present study, little cytoxicity of NaF was observed at lower concentrations, in terms of cell viability, DNA and protein biosynthesis, nucleosomal DNA fragmentation and appearance of hypodiploid cells. In fact, we observed a slight increase in both DNA and protein biosynthesis at concentrations of NaFB50 ppm. It has been reported that exposure to NaF at concentrations ranging from 10 − 6 to 10 − 3 M increases DNA and protein biosynthesis in bone forming cells (Farley et al., 1983) and in rat osteoblasts in culture (Li and DenBestin, 1993). Although these concentrations are lower than those used in this study, it seems that at relatively low levels, NaF has a stimulatory effect on DNA and protein biosynthesis. Our present investigation clearly demonstrates that NaF treatment at 100–200 ppm inhibits cell proliferation and induces apoptosis in human promyelocytic leukemia HL-60 cells. Alterations in cell morphology, fragmentation of nucleosomal DNA, and the appearance of hypodiploid cells all indicate that NaF induces apoptosis in these cells. Apoptosis is a tightly regulated process, which involves changes in the expression of distinct genes. Association of fluoride cytotoxicity and apoptosis induction was suggested (Hirano and Ando, 1996, 1997). Our results of activation of caspase-3 in fluoride-treated cells are in accord with the previous report (Anuradha et al., 2000). It seems that the activation of caspase-3 results from the decrease in Bcl-2 level in fluoride treated cells. The proto-oncogene bcl-2 en-
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Fig. 6. Flow-cytometry analysis of NaF-treated HL-60 cells. (A) Cells were treated with various concentrations of NaF, as indicated, for 24 h. (B) Cells were treated with 200 ppm of NaF for the indicated periods of time. The DNA content of cells in each sample was determined by flow cytometric analysis.
codes a 26 kDa mitochondrial-associated protein that is one of the major genes involved in regulation of apoptosis. The bcl-2 gene product is an intracellular suppressor of apoptosis and thus serves a cyto-protective function in cells (Hengartner and Horvitz, 1994). Furthermore, bcl-2 -transfected cells are often rescued from apoptotic death (Hockenbery, 1995). Our results indicate that NaF treatment (up to 72 h) decreases Bcl-2 immunoreactivity levels in HL-60 cells. The induction of apoptosis by NaF may be caused by this reduction in Bcl-2 levels. In addition, the cleavage of caspase-3 appears to correlate with NaF induced apoptosis in HL-60 cells. Caspase-3 is a cysteine protease that exists as an inactive zymogen in cells. Caspase-3 becomes
activated by sequential proteolytic events that cleave the 32 kDa precursor at aspartic acid residues to generate an active heterodimer comprised of 20 and 12 kDa subunits (Nicholson et al., 1995). The activity of caspase-3 was confirmed by the cleavage of PARP. In summary, NaF, one of the most widely used fluoride compounds, revealed no significant cytotoxicity at lower doses. However, at relatively high doses, NaF induced apoptosis in HL-60 cells. Our data suggest that apoptosis was induced in these cells by a decrease in Bcl-2 immunoreactivity levels and a concomitant activation of caspase-3. Taken together, these findings provide important new insights into the possible molecular mechanisms by which NaF mediates cytotoxicity and apoptosis.
Acknowledgements This study has been carried out under the University Research Institute grant (98-005-F00208) from Korea Research Foundation. J.-S. Song is a graduate fellow and E. Lee is a post-doctoral fellow of Brain Korea 21 program of Ministry of Education, Korea. Fig. 7. Regulation of Bcl-2, caspase-3, and PARP immunoreactivity levels by NaF. HL-60 cells were treated with 200 ppm of NaF for the indicated periods of time. Cells were lysed, and samples containing equal amounts of protein were subjected to immunoblotting with anti-Bcl-2, anti-caspase-3, or anti-PARP antibodies, as described in Section 2.
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