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Antiproliferative effect of a vitamin D3 analog, EB1089, on HL-60 cells by the induction of TGF-β receptor
Leukemia Research 23 (1999) 1105 – 1112 www.elsevier.com/locate/leukres
Antiproliferative effect of a vitamin D3 analog, EB1089, on HL-60 cells by the induction of TGF-b receptor Chul W. Jung a, Eun S. Kim b, Jae G. Seol b, Woo H. Park b, Sang J. Lee a, Byoung K. Kim b, Young Y. Lee b,c,* a Department of Internal Medicine, Chung-Ang Uni6ersity College of Medicine, Seoul 156 -756, South Korea Department of Internal Medicine, Cancer Research Center, Seoul National Uni6ersity College of Medicine, Seoul 110 -799, South Korea c Di6ision of Hematology/Oncology, Department of Internal Medicine, Han Yang Uni6ersity Hospital, 17 Haeng Dang-dong, Sung Dong-ku, Seoul 133 -792, South Korea b
Received 22 January 1999; accepted 28 June 1999
Abstract EB1089 is a novel 1,25(OH)2D3 analog that has more potent antitumor properties with reduced hypercalcemic effects than 1,25(OH)2D3. We investigated the role of transforming growth factor-b1 (TGF-b1) in the growth inhibition of human acute myeloid leukemia cell line, HL-60, by EB1089. Clonal growth of HL-60 cells was inhibited in a dose-dependent manner by EB1089. Although TGF-b1 alone slightly inhibited proliferation of HL-60 cells, the addition of TGF-b1 into culture treated with 10 − 8 M of EB1089 showed a significant synergistic antiproliferative effect in a dose-dependent manner. EB1089 up-regulated the expression of TGF-b receptor type I (TGF-b RI), type II (TGF-b RII) and TGF-b1. Antiproliferative effect of EB1089 was partially reversed by TGF-b1 neutralizing antibody (anti-TGF-b1). Vitamin D receptor (VDR) expression was increased by TGF-b1, suggesting synergistic action of TGF-b1 and EB1089. Combined treatment of EB1089 and TGF-b1 resulted in an increased expression of cyclin-dependent kinase inhibitor (CDKI), p27 protein, compared to either ligand alone. Up-regulation of p27 protein expression by either TGF-b1 or EB1089 was reduced by anti-TGF-b1. These findings suggest that TGF-b1 is involved in the antiproliferative effect of EB1089 on HL-60 cells. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: EB1089; HL-60; Transforming growth factor-b1; Transforming growth factor-b receptor; Vitamin D receptor; p27
1. Introduction The seco-steroid 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] can induce differentiation and inhibit clonal proliferation of acute myelogenous leukemia (AML) cells by binding to a nuclear receptor, vitamin D receptor (VDR) [1,2]. However, therapeutic trials of 1,25(OH)2D3 in leukemia have been restricted by the risk of development of hypercalcemia [3 – 5]. A novel Abbre6iations: AML, acute myelogenous leukemia; Anti-TGF-b1, TGF-b1 neutralizing antibody; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; 1,25 (OH)2D3, 1,25 dihydroxyvitamin D3; TGF, transforming growth factor; TGF-b RI, TGF-b receptor type I; TGF-b RII, TGF-b receptor type II; VDR, vitamin D receptor. * Corresponding author. Tel.: + 82-2-2290-8334; fax: + 82-2-22989183. E-mail address: [email protected] (Y.Y. Lee)
vitamin D3 analog, EB1089, has been shown to be very potent in the inhibition of clonal proliferation of malignant cells including AML cells with reduced hypercalcemic effect [6–8]. The precise mechanism by which 1,25(OH)2D3 compounds exert its biological action on malignant cells remains unclear. In leukemic cells, 1,25(OH)2D3 was shown to accumulate the cyclin-dependent kinase inhibitor (CDKI), p21 or p27, causing G1 arrest [9,10]. Transforming growth factor-b (TGF-b) is a multifunctional cytokine that regulates the proliferation, phenotype, and differentiation of a variety of cell types [11,12]. There are three types of TGF-b; TGF-b1, TGFb2 and TGF-b3, the most commonly studied being TGF-b1 [13]. Depending on the differentiation stage of target cells, TGF-b1 can stimulate or inhibit the proliferation of hematopoietic progenitors. TGF-b1 has shown to be a potent inhibitor of primitive myeloid and
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erythroid progenitors, whereas it has little or no effect on more committed cells [14,15]. Antiproliferative effect of TGF-b1 is mediated via apoptosis or G1 cell cycle arrest resulting from induction of CDKI and suppression of cyclin/cyclin-dependent kinase (CDK) complex [16 – 18]. Recently, evidence has been accumulated that the antiproliferative effects of a number of agents are related to TGFb1. Retinoic acid is known to induce growth inhibition of a promyelocytic leukemic cell line through the induction of TGF-b receptor and TGF-b1 protein, indicating possible TGF-b1-mediated autocrine/paracrine inhibition [19–22]. Likewise, 1,25(OH)2D3 showed antiproliferative effects on breast carcinoma cells and human keratinocytes by the induction of TGF-b1 mRNA expression and secretion of TGF-b1 protein [23,24]. In addition, it has been found that the treatment with TGF-b1 resulted in enhanced sensitivity to the antiproliferative and differentiating effects of 1,25(OH)2D3 in human leukemic cell lines [25 – 27]. However, the mechanism of synergistic antiproliferative effect of TGF-b1 and 1,25(OH)2D3 in leukemic cells is not known. In this study, we investigated the involvement of TGF-b1 in the antiproliferative effect of EB1089 in HL-60 cells. Our results showed that EB1089 enhanced the expression of TGF-b receptors and TGF-b1 induced the expression of VDR, suggesting a potential role of TGF-b1 in autocrine and paracrine growth regulation in EB1089-treated HL-60 cells.
2. Materials and methods
2.1. Cells and cell culture Human myeloid leukemia cell line HL-60 was cultured in tissue flasks in RPMI 1640 (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% (vol/ vol) heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma Chemical, St Louis, MO, USA). All experiments in this study were performed in media containing 10% FBS except growth suppression effect of TGF-b1 in which we used RPMI 1640 containing 2.5% FBS. Cells were maintained in a humidified atmosphere, 5% CO2 at 37°C, culture medium was changed every 3 – 4 days.
2.2. Vitamin D3 compounds, TGF-b1 and TGF-b1 neutralizing antibody The vitamin D3 compounds used in this study were 1,25(OH)2D3 and EB1089 that was a generous gift from Lise Binderup (Leo Pharmaceutical Products, Ballerup, Denmark). Each was dissolved in absolute ethanol at 4×10 − 3 M as a stock solution, stored at − 20°C and
protected from light. Dilutions of the stock solution were made in RPMI 1640 without FBS. The maximum concentration of ethanol in the culture (0.1%) did not influence clonal growth of HL-60 cells. Porcine TGF-b1 (R&D Systems, Minneapolis, MN, USA) which shares complete amino acid homology with human TGF-b1 was used to examine growth suppression with concentrations of 0.1, 1, 2.5, 5 and 10 ng/ml. Neutralizing antibody against TGF-b1 (anti-TGF-b1) used in this study was a pan-specific antibody (R&D Systems) that can neutralize TGF-b1, b2, b3, and b5. We used antiTGF-b1 with the concentration of 50 mg/ml because 5 mg/ml of the antibody can neutralize 50% of 0.25 ng/ml TGF-b1. As a positive control for the activity of antiTGF-b1, we used acute myeloid leukemia cell line (U937) that was known to be sensitive to growth suppression by EB1089 and TGF-b1 [28]. Anti-TGF-b1 reversed EB1089-induced growth suppression in U937 from 34 to 60% of control.
2.3. Clonogenic assay and [ 3H]thymidine incorporation assay HL-60 cells were plated in tissue culture grade 35mm petri dish in a volume of 1 ml RPMI containing 10% FBS, 10% bovine serum albumin (BSA; Sigma), 1.2% methylcellulose (Eastman Kodak, Rochester, NY, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were plated at 2×103 per dish, incubated in a humidified atmosphere, 5% CO2 at 37°C for 6–10 days. Colonies ( \ 40 cells) were scored with an inverted microscope. Growth inhibitory effect of TGF-b1 was assessed by the [3H]thymidine incorporation. HL-60 cells (1 × 104) were suspended in 200 ml of RPMI 1640 with 2.5% FBS, and placed in wells of a 96-well microtiter plate (Nunc, Naperville, IL, USA) in the presence or absence of TGF-b1. After exposure to TGF-b1 for 4 days, 0.5 mCi of [3H]thymidine (ICN, Costa Mesa, CA, USA) was added to each well, and cells were incubated for additional 4 h at 37°C. The medium was removed, and the samples were harvested onto glass fiber filters, washed, and analyzed for [3H]thymidine incorporation by liquid scintillation counting. Relative radioactivity of each well was depicted as the percentage of that of the untreated control.
2.4. TGF-b1 assay To measure active TGF-b1, HL-60 (2 × 103 cells) were cultured in 35-mm petri dish in the presence and absence of 1× 10 − 8 M of EB1089. Supernatants were harvested after various times of exposure to EB1089. Acid-activated (according to the manufacturer’s protocol) supernatant samples were assayed for biologically active TGF-b1 using a commercially available ELISA kit (QuantikineTM, R&D). This method can detect up to 5 pg/ml of TGF-b1.
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2.5. Northern blot analysis Cultured cells were harvested, washed with phosphate-buffered saline (PBS), and stored at − 70°C until use. Total RNA was extracted by the TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA). A volume of 15 mg of total RNA per sample was size-fractionated through 1% denaturating formaldehyde agarose gel and transferred on Nytran membrane (Schleicher and Schuell, Keene, NH). Blots were prehybridized with 7% sodium dodecyl sulfate (SDS)/0.5 M sodium phosphate/1 mM EDTA at 42°C for 4 h, and hybridization was done using a-[32P]dCTP-labeled cDNA probes of TGF-b1, TGF-b type I and type II receptor, and VDR. After hybridization, the membranes were washed, and exposed to Kodak XAR 5 film (Eastman Kodak) at −70°C for 24 h. The filters were reprobed with b-actin cDNA to normalize for RNA loading.
2.6. Western blot analysis Cells were washed with PBS, suspended in lysis buffer containing 50 mM Tris (pH 7.5), 1% NP-40, 2 mM EDTA, 10 mM NaCl, 20 mg/ml aprotinin, 20 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and placed on ice for 30 min. After centrifugation for 1 h at 4°C at 15 000 × g, the supernatant was collected. Whole lysate (100 mg) was resolved by 12% SDS polyacrylamide gel, transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) by electroblotting, and probed with various monoclonal antibodies (Transduction Laboratory, Lexington, KY, USA). The blot was developed by using the ECL kit (Amersham, Arlington Heights, IL, USA).
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Then, we assessed the effect of TGF-b1 on HL-60 cells by [3H]thymidine incorporation assay. TGF-b1 alone had little effect on proliferation of HL-60 cells, although treatment of HL-60 cells with increasing concentrations of TGF-b1 resulted in a slight decrease in proliferation (data not shown). However, addition of TGF-b1 ranging from 0.1 to 10 ng/ml into HL-60 cells treated with 1,25(OH)2D3 (1×10 − 8 M) revealed a marked inhibition of proliferation in a dose-dependent manner (Fig. 2).
3.2. Induction of expression of TGF-b receptors and TGF-b1 by EB1089 Since TGF-b1 had a synergistic effect on proliferation of HL-60 cells when combined with EB1089, we examined whether EB1089 could induce the expression of TGF-b receptors. Treatment with 10 − 8 M of EB1089 for 72 h enhanced mRNA and protein expression of TGF-b receptor type I (TGF-b RI) and TGF-b receptor type II (TGF-b RII) (Fig. 3). Neither structural changes nor amplifications of TGF-b RI and TGF-b RII genes were seen in HL-60 cells by Southern blot analysis (data not shown). Since EB1089 increased TGF-b receptor expression and resulted in an enhanced sensitivity to the antiproliferative effects of TGF-b1, we wanted to determine whether EB1089 could induce enhanced transcription and secretion of TGF-b1. It was found that 10 − 8 M of EB1089 enhanced the expression of TGF-b1 mRNA in a time-dependent manner since 48 h of treatment (Fig. 4A). Active TGF-b1 protein secretion also increased in accordance with TGF-b1
3. Results
3.1. Effect of 1,25(OH)2D3, EB1089 and TGF-b1 on clonal growth of HL-60 cells We examined the effect of 1,25(OH)2D3 and EB1089 on clonal proliferation of a human myeloid leukemia cell line, HL-60. Both compounds exhibited significant inhibition of cell growth of HL-60 in a dose-dependent manner (Fig. 1). The EB1089 at 1 × 10 − 10 M showed greater than 50% inhibition of clonal growth of HL-60 cells, the calculated concentration that inhibited 50% growth (ED50) could not be achieved through the concentrations used in this experiment. In contrast, the reference compound, 1,25(OH)2D3, produced an ED50 of about 1.3×10 − 8 M, demonstrating that EB1089 is more potent in inhibiting the proliferation of HL-60 cells than 1,25(OH)2D3. These vitamin D3 compounds induced apoptosis in HL-60 cells (data not shown).
Fig. 1. Dose-response of vitamin D3 compounds, 1,25(OH)2D3 and EB1089 on clonal growth of HL-60 cells. HL-60 cells were plated in methylcellulose with and without vitamin D3 compounds at various concentrations. Colonies were enumerated after 6 – 10 days of culture. Results are expressed as a percent of control plates not exposed to vitamin D3 compounds. Each point represents the mean of three experiments with triplicate dishes. Bar represents standard error. ED50, effective dose achieving 50% response; NR, ED50 could not be achieved.
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Fig. 2. Synergistic growth inhibition by EB1089 and TGF-b1. HL-60 cells (1 × 104) treated with 10 − 8 M of EB1089 were replated in 96-well microtiter plate in the absence or presence of TGF-b1 at various concentrations. Cultures were incubated at 37°C for 4 days under low serum conditions (2.5%), and pulsed with 0.5 mCi of [3H]thymidine per well for additional 4 h. The amount of thymidine incorporated was determined by liquid scintillation counting. Results represent the mean of at least three independent experiments with six replicate determinations.
mRNA induction. At 72 h of culture, concentration of TGF-b1 protein in the supernatants by ELISA markedly increased up to 140 pg/ml (Fig. 4B). Although 140 pg/ml of exogenously treated TGF- b1 is not enough to be inhibitory, it may exert an antiprolif-
Fig. 3. Expression of TGF-b1 receptors by EB1089-treated HL-60 cells. Total RNA from HL-60 cells treated with 10 − 8 M of EB1089 for 72 h was transferred to Nytran membrane after denaturing formaldehyde gel electrophoresis and hybridized with cDNA probes of TGF-b receptor type I (TGF-b RI) (A, upper) and type II (TGF-b RII) (B, upper). As a control, all blots were rehybridized with b-actin cDNA. Transcriptions of mRNA of TGF-b RI and RII were enhanced by 72-h treatment with EB1089. Western blot was prepared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) after cell lysis with detergent. After transfer to nitrocellulose membrane, immunoblotting was accomplished with a monoclonal anti-TGF-b RI antibody (A, lower) and a monoclonal anti-TGF-b RII antibody (B, lower). A volume of 10 − 8 M of EB1089 treatment for 72 h induced expression of TGF-b RI protein and TGF-b RII protein. Tubulin was used as a control.
Fig. 4. Induction of TGF-b1 mRNA and protein secretion in EB1089-treated HL-60 cells. Total RNAs from HL-60 cells treated with EB1089 (10 − 8 M) for indicated times were transferred to Nytran membrane after denaturing formaldehyde gel electrophoresis and hybridized with cDNA probe of TGF-b1. As a control, the filter was reprobed with b-actin cDNA to normalize for RNA loading (A). TGF-b1 in the supernatants of HL-60 stimulated by EB1089 (10 − 8 M) was measured with ELISA. Active TGF-b1 protein secretion markedly increased at 72 h in accordance with the induction of RNA by EB1089. As a control, cells were cultured without EB1089 (B).
erative effect on HL-60 cells if enhanced expression of TGF-b RI and RII was considered.
3.3. Effect of neutralizing antibody against TGF-b1 on EB1089 -treated HL-60 cells Since EB1089 increased the expression of TGF-b receptors and TGF-b1 in HL-60 cells, we investigated whether TGF-b1 neutralizing antibody (anti-TGF-b1) could abrogate the antiproliferative effect of EB1089 by clonogenic assay. Anti-TGF-b1 had little effect on clonal proliferation of HL-60 cells by semisolid culture. EB1089 (10 − 8 M) alone inhibited clonal proliferation of HL-60 cells to 22% of controls. This growth inhibition of HL-60 cells by EB1089 was partially reversed to 45% of controls by 50 mg/ml of anti-TGF-b1 (Fig. 5). This result suggests that TGF-b1 may be involved in the antiproliferative effect of EB1089 on HL-60.
3.4. Induction of 6itamin D receptor by TGF-b1 Vitamin D3 compounds exert their biologic action through binding with a specific intracellular receptor, vitamin D receptor (VDR). To further elucidate the synergistic mechanism of TGF-b1 and EB1089, the expression of VDR mRNA in TGF-b1-treated HL-60 cells was assessed at various times of treatment. The concentration of TGF-b1 used in this experiment was 2.5 ng/ml, because higher concentrations of TGF-b1 did not result in a further significant decrease in prolif-
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Fig. 5. Effect of TGF-b1 neutralizing antibody (anti-TGF-b1) on EB1089-induced inhibition of HL-60. Cells were plated at 2 ×103 cells per dish in the presence of EB1089 (10 − 8 M) alone or combination of EB1089 and anti-TGF-b1 (50 mg/ml). Colonies were counted after 7 days of culture. Results are expressed as the mean percentages of control plates, and each value represents the mean of three experiments with standard deviations.
Fig. 7. Expression of p27 protein by EB1089 and TGF-b1. After 72 h culture of HL-60 with EB1089 (10 − 8 M), TGF-b1 (2.5 ng/ml), anti-TGF-b1 (50 mg/ml), or combinations of these agents, 100 mg of total lysates was resolved by 12% SDS polyacrylamide gel, transferred onto a nitrocellulose membrane, and probed with anti-p27 monoclonal antibody. EB1089 and TGF-b1 synergistically induced p27 protein compared to EB1089 or TGF-b1 alone. Anti-TGF-b1 partially blocked induction of p27 protein by EB1089 or TGF-b1 alone. Tubulin was used as a loading control. The p27 protein levels were quantitated using a densitometer and expressed in arbitrary units. CTR, control; EB, EB1089; T, TGF-b1; Ab, anti-TGF-b1.
eration (Fig. 2). The up-regulation of VDR mRNA was demonstrated with a peak at 4 h of exposure to TGFb1 and returned to the pretreatment level at 72 h (Fig. 6A). Western blot analysis using an anti-VDR mono-
clonal antibody showed that VDR protein expression was also up-regulated by TGF-b1 in accordance with VDR mRNA induction and the induction was apparent after 48 h of TGF-b1 treatment (Fig. 6B).
3.5. Expression of p27 by EB1089 and TGF-b1 Since cell cycle arrest by TGF-b1 is known to be mediated by p27, we examined the change of p27 protein expression in HL-60 cells at 72 h after treatment of EB1089 (10 − 8 M) and TGF-b1 (2.5 ng/ml). Both EB1089 and TGF-b1 enhanced the expression of p27 protein (Fig. 7). Signal intensity of p27 in EB1089treated HL-60 cells was stronger than TGF-b1, which was consistent in our antiproliferative data. Addition of anti-TGF-b1 to EB1089-treated HL-60 cells or TGFb1-treated HL-60 cells resulted in relatively decreased expression of p27 compared to EB1089 or TGF-b1 alone, respectively. Combined treatment of EB1089 and TGF-b1 synergistically increased the induction of p27 expression compared to either ligand alone. Fig. 6. Enhanced expression of VDR by TGF-b1. Total RNAs from HL-60 cells treated with TGF-b1 (2.5 ng/ml) for indicated times were transferred to Nytran membrane after denaturing formaldehyde gel electrophoresis and hybridized with cDNA probe of VDR. As a control, the filter was rehybridized with b-actin cDNA (A). Western blot was prepared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after cell lysis with detergent. After transfer to nitrocellulose membrane, immunoblotting was accomplished with a monoclonal anti-VDR antibody. TGF-b1 induced expression of VDR protein after 48 h of treatment. Tubulin was used as a control (B).
4. Discussion In the present study, we investigated the role of TGF-b1 in antiproliferative effect by 1,25(OH)2D3 analog, EB1089, which has more significant antitumoral activities than its parental compound, 1,25(OH)2D3. Consistent with previous study, EB1089 showed a marked growth inhibition of HL-60 cells compared to 1,25(OH)2D3 [8]. Although the precise
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antiproliferative mechanism of this vitamin D3 analog is unknown, it may be related to low affinity of vitamin D3 analog to plasma transport protein and more avid binding to its receptor [29] or to an enhanced dimerization of vitamin D receptor (VDR) with retinoic acid X receptor (RXR) compared to 1,25(OH)2D3 [30]. Acute myelogenous leukemia (AML) cells show heterogeneous response to TGF-b1 [31 – 33]. In our experiments, TGF-b1 alone had minor effect on proliferation of HL-60 cells, which is consistent with other reports [34,35]. However, combined treatment of TGF-b1 and EB1089 resulted in a significant inhibition compared to either ligand alone. The mechanism by which TGF-b1 renders HL-60 cells more sensitive to the antiproliferative activity of 1,25(OH)2D3 compound is unknown. Several lines of evidence suggest that the antiproliferative effects of several agents on malignant cells are associated with TGF-b1 [19 – 22,35]. In human osteoblasts, 1,25(OH)2D3 increased expression of TGF-b1 and TGF-b receptors [36,37]. Very recently, a positive regulation by 1,25(OH)2D3 of TGF –b1-mediated AP-1 activity was suggested in osteoblastic cells [38]. Therefore, one possible mechanism of increased antiproliferative sensitivity by EB1089 of TGF-b1 may be modulation of TGF-b1 and TGF-b1 receptor expressions. Treatment of HL-60 cells with EB1089 up-regulated the expression of TGF-b1 and TGF-b receptors, suggesting a role of TGF-b1 in antiproliferative effect of EB1089. Furthermore, to determine whether TGFb1 is involved in EB1089-induced antiproliferative effect, TGF-b1 neutralizing antibody (anti-TGF-b1) was added into HL-60 cells simultaneously treated with TGF-b1 and EB1089. Although anti-TGF-b1 can effectively neutralize endogenous TGF-b1 only under serum-free condition [39,40], we examined it under serum-containing condition, because it is difficult to culture HL-60 cells in serum-free condition, and because 1,25(OH)2D3 analogs have been shown to be more potent in the presence of serum [29]. Antiproliferative effect of EB1089 was partially blocked by antiTGF-b1. Thus, autocrine secretion of TGF-b1 may be a significant antiproliferative mechanism of EB1089 in HL-60 cells. VDR is a nuclear protein that mediates the biological actions of 1,25(OH)2D3 analogs and its level is believed to determine the cellular responsiveness [41 – 43]. In this study, we found that expression of VDR mRNA was elevated at 4 h following treatment of TGF-b1 and returned to the pretreatment level at 72 h. It is consistent with the previous reports that up-regulation of VDR mRNA in HL-60 cells by 1,25(OH)2D3 reached a peak at 1 or 2 h [41,44]. Similarly, expression of VDR protein was increased by TGF-b1, which was apparent at 48 h. Collectively, these experiments demonstrated that synergistic antiproliferative effects of EB1089 and TGF-b1 came from the enhanced expression of TGF-
b1 by EB1089 and the increased expression of VDR by TGF-b1. It has been suggested that target molecule involved in cell cycle arrest by vitamin D3 compounds is cyclin-dependent kinase inhibitor (CDKI), p21 or p27 [9,10]. However, p21 expression by 1,25(OH)2D3 was reported to be only transient and did not correlate with the onset of the G1 block in HL-60 cells [45]. Recently, Wang et al. [46] demonstrated that p27 induced G1 arrest in HL-60 cells that was reversed by an oligonucleotide antisense to p27. Asou et al. [47] reported that antiproliferative effect by a vitamin D3 analog, Ro25-9716, was associated with the induction of p27 in HL-60 cells. Therefore, it is likely that p27 is a principal mediator in vitamin D3 compound-induced cell cycle arrest. We also found that EB1089 increased the induction of p27 protein in a time- and a dose-dependent manner (data not shown). Combined treatment with TGF-b1 and EB1089 markedly increased the expression of p27 protein compared to either ligand alone, and the induction of p27 protein by TGF-b1 or EB1089 was partially blocked by anti-TGF-b1. Since p27 is known to mediate TGF-b1-induced G1 arrest [48,49], these results provide an additional evidence that TGF-b1 mediates antiproliferative effect of EB1089. In summary, EB1089 was more potent in the growth suppression of HL-60 cells than 1,25(OH)2D3. EB1089induced growth suppression was associated with the induction of TGF-b1, TGF-b RI, and TGF-b RII, and was partially reversed by the addition of anti-TGF-b1. TGF-b1 itself enhanced the expression of VDR, that explained the synergistic antiproliferative effect of TGF-b1 and EB1089. Anti-TGF-b1 partially blocked the induction of p27 by EB1089 and TGF-b1. These results suggest that the antiproliferative effect of EB1089 is mediated by TGF-b1 through p27 in HL-60 cells.
Acknowledgements This study was supported by a grant of the 1997 Korean Nation Cancer Control Program, Ministry of Health and Welfare, R.O.K. CW Jung contributed to the concept and design, drafted the manuscript, provided significant statistical expertise, administrative support and critical revision of the paper. ES Kim helped with the data collection, provided technical support, statistical assistance and interpretation of the data. JG Seol provided technical support and helped with data collection, assisted in drafting the paper and in data analysis. WH Park provided technical assistance and data collection, helped with biostatistics, analysis of data and drafting the paper. SJ Lee provided some of the funding, provided study materials and critical review of the paper. BK Kim provided some of the
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funds, study materials and gave critical revision. YY Lee contributed in a major way to the study concept, gave final approval, helped with data analysis, assembly of data, administrative support, partial funding of the project and provided critical revision of the paper.
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[43] Kanatani Y, Makishima Y, Ishikawa I, Ogasawara Y, Kawahara N, Motoyoshi K, et al. A novel uracil analog, 6-chloro-5-(2propenyl)uracil, preferentially enhances growth inhibition and differentiation of myeloid leukemia cells induced by 1alpha,25-dihydroxyvitamin D3. Exp Hematol 1998;26:198. [44] Taoka T, Collins ED, Irino S, Norman AW. 1,25(OH)2-vitamin D3 mediated changes in mRNA for c-myc and 1,25(OH)2D3 receptor in HL-60 cells and related subclones. Mol Cell Endocrinol 1993;95:51. [45] Wang QM, Jones JB, Studzinski GP. Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells. Cancer Res 1996;56:264. [46] Wang QM, Chen F, Luo X, Moore DC, Flanagan M, Studzinski GP. Lowering of p27Kip1 levels by its antisense or by development of resistance to 1,25-dihydroxyvitamin D3 reverses the G1 block but not differentiation of HL60 cells. Leukemia 1998;12:1256. [47] Asou H, Koike M, Elstner E, Cambell M, Le J, Uskokovic MR, et al. 19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines. Blood 1998;92:2441. [48] Polyak K, Lee MH, Erdjument-Bromaga H, Koff A, Roberts JM, Tempst P, et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitotic signals. Cell 1994;78:59. [49] Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 1994;78:67.
Antiproliferative effect of a vitamin D3 analog, EB1089, on HL-60 cells by the induction of TGF-b receptor Chul W. Jung a, Eun S. Kim b, Jae G. Seol b, Woo H. Park b, Sang J. Lee a, Byoung K. Kim b, Young Y. Lee b,c,* a Department of Internal Medicine, Chung-Ang Uni6ersity College of Medicine, Seoul 156 -756, South Korea Department of Internal Medicine, Cancer Research Center, Seoul National Uni6ersity College of Medicine, Seoul 110 -799, South Korea c Di6ision of Hematology/Oncology, Department of Internal Medicine, Han Yang Uni6ersity Hospital, 17 Haeng Dang-dong, Sung Dong-ku, Seoul 133 -792, South Korea b
Received 22 January 1999; accepted 28 June 1999
Abstract EB1089 is a novel 1,25(OH)2D3 analog that has more potent antitumor properties with reduced hypercalcemic effects than 1,25(OH)2D3. We investigated the role of transforming growth factor-b1 (TGF-b1) in the growth inhibition of human acute myeloid leukemia cell line, HL-60, by EB1089. Clonal growth of HL-60 cells was inhibited in a dose-dependent manner by EB1089. Although TGF-b1 alone slightly inhibited proliferation of HL-60 cells, the addition of TGF-b1 into culture treated with 10 − 8 M of EB1089 showed a significant synergistic antiproliferative effect in a dose-dependent manner. EB1089 up-regulated the expression of TGF-b receptor type I (TGF-b RI), type II (TGF-b RII) and TGF-b1. Antiproliferative effect of EB1089 was partially reversed by TGF-b1 neutralizing antibody (anti-TGF-b1). Vitamin D receptor (VDR) expression was increased by TGF-b1, suggesting synergistic action of TGF-b1 and EB1089. Combined treatment of EB1089 and TGF-b1 resulted in an increased expression of cyclin-dependent kinase inhibitor (CDKI), p27 protein, compared to either ligand alone. Up-regulation of p27 protein expression by either TGF-b1 or EB1089 was reduced by anti-TGF-b1. These findings suggest that TGF-b1 is involved in the antiproliferative effect of EB1089 on HL-60 cells. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: EB1089; HL-60; Transforming growth factor-b1; Transforming growth factor-b receptor; Vitamin D receptor; p27
1. Introduction The seco-steroid 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] can induce differentiation and inhibit clonal proliferation of acute myelogenous leukemia (AML) cells by binding to a nuclear receptor, vitamin D receptor (VDR) [1,2]. However, therapeutic trials of 1,25(OH)2D3 in leukemia have been restricted by the risk of development of hypercalcemia [3 – 5]. A novel Abbre6iations: AML, acute myelogenous leukemia; Anti-TGF-b1, TGF-b1 neutralizing antibody; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; 1,25 (OH)2D3, 1,25 dihydroxyvitamin D3; TGF, transforming growth factor; TGF-b RI, TGF-b receptor type I; TGF-b RII, TGF-b receptor type II; VDR, vitamin D receptor. * Corresponding author. Tel.: + 82-2-2290-8334; fax: + 82-2-22989183. E-mail address: [email protected] (Y.Y. Lee)
vitamin D3 analog, EB1089, has been shown to be very potent in the inhibition of clonal proliferation of malignant cells including AML cells with reduced hypercalcemic effect [6–8]. The precise mechanism by which 1,25(OH)2D3 compounds exert its biological action on malignant cells remains unclear. In leukemic cells, 1,25(OH)2D3 was shown to accumulate the cyclin-dependent kinase inhibitor (CDKI), p21 or p27, causing G1 arrest [9,10]. Transforming growth factor-b (TGF-b) is a multifunctional cytokine that regulates the proliferation, phenotype, and differentiation of a variety of cell types [11,12]. There are three types of TGF-b; TGF-b1, TGFb2 and TGF-b3, the most commonly studied being TGF-b1 [13]. Depending on the differentiation stage of target cells, TGF-b1 can stimulate or inhibit the proliferation of hematopoietic progenitors. TGF-b1 has shown to be a potent inhibitor of primitive myeloid and
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erythroid progenitors, whereas it has little or no effect on more committed cells [14,15]. Antiproliferative effect of TGF-b1 is mediated via apoptosis or G1 cell cycle arrest resulting from induction of CDKI and suppression of cyclin/cyclin-dependent kinase (CDK) complex [16 – 18]. Recently, evidence has been accumulated that the antiproliferative effects of a number of agents are related to TGFb1. Retinoic acid is known to induce growth inhibition of a promyelocytic leukemic cell line through the induction of TGF-b receptor and TGF-b1 protein, indicating possible TGF-b1-mediated autocrine/paracrine inhibition [19–22]. Likewise, 1,25(OH)2D3 showed antiproliferative effects on breast carcinoma cells and human keratinocytes by the induction of TGF-b1 mRNA expression and secretion of TGF-b1 protein [23,24]. In addition, it has been found that the treatment with TGF-b1 resulted in enhanced sensitivity to the antiproliferative and differentiating effects of 1,25(OH)2D3 in human leukemic cell lines [25 – 27]. However, the mechanism of synergistic antiproliferative effect of TGF-b1 and 1,25(OH)2D3 in leukemic cells is not known. In this study, we investigated the involvement of TGF-b1 in the antiproliferative effect of EB1089 in HL-60 cells. Our results showed that EB1089 enhanced the expression of TGF-b receptors and TGF-b1 induced the expression of VDR, suggesting a potential role of TGF-b1 in autocrine and paracrine growth regulation in EB1089-treated HL-60 cells.
2. Materials and methods
2.1. Cells and cell culture Human myeloid leukemia cell line HL-60 was cultured in tissue flasks in RPMI 1640 (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% (vol/ vol) heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma Chemical, St Louis, MO, USA). All experiments in this study were performed in media containing 10% FBS except growth suppression effect of TGF-b1 in which we used RPMI 1640 containing 2.5% FBS. Cells were maintained in a humidified atmosphere, 5% CO2 at 37°C, culture medium was changed every 3 – 4 days.
2.2. Vitamin D3 compounds, TGF-b1 and TGF-b1 neutralizing antibody The vitamin D3 compounds used in this study were 1,25(OH)2D3 and EB1089 that was a generous gift from Lise Binderup (Leo Pharmaceutical Products, Ballerup, Denmark). Each was dissolved in absolute ethanol at 4×10 − 3 M as a stock solution, stored at − 20°C and
protected from light. Dilutions of the stock solution were made in RPMI 1640 without FBS. The maximum concentration of ethanol in the culture (0.1%) did not influence clonal growth of HL-60 cells. Porcine TGF-b1 (R&D Systems, Minneapolis, MN, USA) which shares complete amino acid homology with human TGF-b1 was used to examine growth suppression with concentrations of 0.1, 1, 2.5, 5 and 10 ng/ml. Neutralizing antibody against TGF-b1 (anti-TGF-b1) used in this study was a pan-specific antibody (R&D Systems) that can neutralize TGF-b1, b2, b3, and b5. We used antiTGF-b1 with the concentration of 50 mg/ml because 5 mg/ml of the antibody can neutralize 50% of 0.25 ng/ml TGF-b1. As a positive control for the activity of antiTGF-b1, we used acute myeloid leukemia cell line (U937) that was known to be sensitive to growth suppression by EB1089 and TGF-b1 [28]. Anti-TGF-b1 reversed EB1089-induced growth suppression in U937 from 34 to 60% of control.
2.3. Clonogenic assay and [ 3H]thymidine incorporation assay HL-60 cells were plated in tissue culture grade 35mm petri dish in a volume of 1 ml RPMI containing 10% FBS, 10% bovine serum albumin (BSA; Sigma), 1.2% methylcellulose (Eastman Kodak, Rochester, NY, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were plated at 2×103 per dish, incubated in a humidified atmosphere, 5% CO2 at 37°C for 6–10 days. Colonies ( \ 40 cells) were scored with an inverted microscope. Growth inhibitory effect of TGF-b1 was assessed by the [3H]thymidine incorporation. HL-60 cells (1 × 104) were suspended in 200 ml of RPMI 1640 with 2.5% FBS, and placed in wells of a 96-well microtiter plate (Nunc, Naperville, IL, USA) in the presence or absence of TGF-b1. After exposure to TGF-b1 for 4 days, 0.5 mCi of [3H]thymidine (ICN, Costa Mesa, CA, USA) was added to each well, and cells were incubated for additional 4 h at 37°C. The medium was removed, and the samples were harvested onto glass fiber filters, washed, and analyzed for [3H]thymidine incorporation by liquid scintillation counting. Relative radioactivity of each well was depicted as the percentage of that of the untreated control.
2.4. TGF-b1 assay To measure active TGF-b1, HL-60 (2 × 103 cells) were cultured in 35-mm petri dish in the presence and absence of 1× 10 − 8 M of EB1089. Supernatants were harvested after various times of exposure to EB1089. Acid-activated (according to the manufacturer’s protocol) supernatant samples were assayed for biologically active TGF-b1 using a commercially available ELISA kit (QuantikineTM, R&D). This method can detect up to 5 pg/ml of TGF-b1.
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2.5. Northern blot analysis Cultured cells were harvested, washed with phosphate-buffered saline (PBS), and stored at − 70°C until use. Total RNA was extracted by the TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA). A volume of 15 mg of total RNA per sample was size-fractionated through 1% denaturating formaldehyde agarose gel and transferred on Nytran membrane (Schleicher and Schuell, Keene, NH). Blots were prehybridized with 7% sodium dodecyl sulfate (SDS)/0.5 M sodium phosphate/1 mM EDTA at 42°C for 4 h, and hybridization was done using a-[32P]dCTP-labeled cDNA probes of TGF-b1, TGF-b type I and type II receptor, and VDR. After hybridization, the membranes were washed, and exposed to Kodak XAR 5 film (Eastman Kodak) at −70°C for 24 h. The filters were reprobed with b-actin cDNA to normalize for RNA loading.
2.6. Western blot analysis Cells were washed with PBS, suspended in lysis buffer containing 50 mM Tris (pH 7.5), 1% NP-40, 2 mM EDTA, 10 mM NaCl, 20 mg/ml aprotinin, 20 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and placed on ice for 30 min. After centrifugation for 1 h at 4°C at 15 000 × g, the supernatant was collected. Whole lysate (100 mg) was resolved by 12% SDS polyacrylamide gel, transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) by electroblotting, and probed with various monoclonal antibodies (Transduction Laboratory, Lexington, KY, USA). The blot was developed by using the ECL kit (Amersham, Arlington Heights, IL, USA).
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Then, we assessed the effect of TGF-b1 on HL-60 cells by [3H]thymidine incorporation assay. TGF-b1 alone had little effect on proliferation of HL-60 cells, although treatment of HL-60 cells with increasing concentrations of TGF-b1 resulted in a slight decrease in proliferation (data not shown). However, addition of TGF-b1 ranging from 0.1 to 10 ng/ml into HL-60 cells treated with 1,25(OH)2D3 (1×10 − 8 M) revealed a marked inhibition of proliferation in a dose-dependent manner (Fig. 2).
3.2. Induction of expression of TGF-b receptors and TGF-b1 by EB1089 Since TGF-b1 had a synergistic effect on proliferation of HL-60 cells when combined with EB1089, we examined whether EB1089 could induce the expression of TGF-b receptors. Treatment with 10 − 8 M of EB1089 for 72 h enhanced mRNA and protein expression of TGF-b receptor type I (TGF-b RI) and TGF-b receptor type II (TGF-b RII) (Fig. 3). Neither structural changes nor amplifications of TGF-b RI and TGF-b RII genes were seen in HL-60 cells by Southern blot analysis (data not shown). Since EB1089 increased TGF-b receptor expression and resulted in an enhanced sensitivity to the antiproliferative effects of TGF-b1, we wanted to determine whether EB1089 could induce enhanced transcription and secretion of TGF-b1. It was found that 10 − 8 M of EB1089 enhanced the expression of TGF-b1 mRNA in a time-dependent manner since 48 h of treatment (Fig. 4A). Active TGF-b1 protein secretion also increased in accordance with TGF-b1
3. Results
3.1. Effect of 1,25(OH)2D3, EB1089 and TGF-b1 on clonal growth of HL-60 cells We examined the effect of 1,25(OH)2D3 and EB1089 on clonal proliferation of a human myeloid leukemia cell line, HL-60. Both compounds exhibited significant inhibition of cell growth of HL-60 in a dose-dependent manner (Fig. 1). The EB1089 at 1 × 10 − 10 M showed greater than 50% inhibition of clonal growth of HL-60 cells, the calculated concentration that inhibited 50% growth (ED50) could not be achieved through the concentrations used in this experiment. In contrast, the reference compound, 1,25(OH)2D3, produced an ED50 of about 1.3×10 − 8 M, demonstrating that EB1089 is more potent in inhibiting the proliferation of HL-60 cells than 1,25(OH)2D3. These vitamin D3 compounds induced apoptosis in HL-60 cells (data not shown).
Fig. 1. Dose-response of vitamin D3 compounds, 1,25(OH)2D3 and EB1089 on clonal growth of HL-60 cells. HL-60 cells were plated in methylcellulose with and without vitamin D3 compounds at various concentrations. Colonies were enumerated after 6 – 10 days of culture. Results are expressed as a percent of control plates not exposed to vitamin D3 compounds. Each point represents the mean of three experiments with triplicate dishes. Bar represents standard error. ED50, effective dose achieving 50% response; NR, ED50 could not be achieved.
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Fig. 2. Synergistic growth inhibition by EB1089 and TGF-b1. HL-60 cells (1 × 104) treated with 10 − 8 M of EB1089 were replated in 96-well microtiter plate in the absence or presence of TGF-b1 at various concentrations. Cultures were incubated at 37°C for 4 days under low serum conditions (2.5%), and pulsed with 0.5 mCi of [3H]thymidine per well for additional 4 h. The amount of thymidine incorporated was determined by liquid scintillation counting. Results represent the mean of at least three independent experiments with six replicate determinations.
mRNA induction. At 72 h of culture, concentration of TGF-b1 protein in the supernatants by ELISA markedly increased up to 140 pg/ml (Fig. 4B). Although 140 pg/ml of exogenously treated TGF- b1 is not enough to be inhibitory, it may exert an antiprolif-
Fig. 3. Expression of TGF-b1 receptors by EB1089-treated HL-60 cells. Total RNA from HL-60 cells treated with 10 − 8 M of EB1089 for 72 h was transferred to Nytran membrane after denaturing formaldehyde gel electrophoresis and hybridized with cDNA probes of TGF-b receptor type I (TGF-b RI) (A, upper) and type II (TGF-b RII) (B, upper). As a control, all blots were rehybridized with b-actin cDNA. Transcriptions of mRNA of TGF-b RI and RII were enhanced by 72-h treatment with EB1089. Western blot was prepared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) after cell lysis with detergent. After transfer to nitrocellulose membrane, immunoblotting was accomplished with a monoclonal anti-TGF-b RI antibody (A, lower) and a monoclonal anti-TGF-b RII antibody (B, lower). A volume of 10 − 8 M of EB1089 treatment for 72 h induced expression of TGF-b RI protein and TGF-b RII protein. Tubulin was used as a control.
Fig. 4. Induction of TGF-b1 mRNA and protein secretion in EB1089-treated HL-60 cells. Total RNAs from HL-60 cells treated with EB1089 (10 − 8 M) for indicated times were transferred to Nytran membrane after denaturing formaldehyde gel electrophoresis and hybridized with cDNA probe of TGF-b1. As a control, the filter was reprobed with b-actin cDNA to normalize for RNA loading (A). TGF-b1 in the supernatants of HL-60 stimulated by EB1089 (10 − 8 M) was measured with ELISA. Active TGF-b1 protein secretion markedly increased at 72 h in accordance with the induction of RNA by EB1089. As a control, cells were cultured without EB1089 (B).
erative effect on HL-60 cells if enhanced expression of TGF-b RI and RII was considered.
3.3. Effect of neutralizing antibody against TGF-b1 on EB1089 -treated HL-60 cells Since EB1089 increased the expression of TGF-b receptors and TGF-b1 in HL-60 cells, we investigated whether TGF-b1 neutralizing antibody (anti-TGF-b1) could abrogate the antiproliferative effect of EB1089 by clonogenic assay. Anti-TGF-b1 had little effect on clonal proliferation of HL-60 cells by semisolid culture. EB1089 (10 − 8 M) alone inhibited clonal proliferation of HL-60 cells to 22% of controls. This growth inhibition of HL-60 cells by EB1089 was partially reversed to 45% of controls by 50 mg/ml of anti-TGF-b1 (Fig. 5). This result suggests that TGF-b1 may be involved in the antiproliferative effect of EB1089 on HL-60.
3.4. Induction of 6itamin D receptor by TGF-b1 Vitamin D3 compounds exert their biologic action through binding with a specific intracellular receptor, vitamin D receptor (VDR). To further elucidate the synergistic mechanism of TGF-b1 and EB1089, the expression of VDR mRNA in TGF-b1-treated HL-60 cells was assessed at various times of treatment. The concentration of TGF-b1 used in this experiment was 2.5 ng/ml, because higher concentrations of TGF-b1 did not result in a further significant decrease in prolif-
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Fig. 5. Effect of TGF-b1 neutralizing antibody (anti-TGF-b1) on EB1089-induced inhibition of HL-60. Cells were plated at 2 ×103 cells per dish in the presence of EB1089 (10 − 8 M) alone or combination of EB1089 and anti-TGF-b1 (50 mg/ml). Colonies were counted after 7 days of culture. Results are expressed as the mean percentages of control plates, and each value represents the mean of three experiments with standard deviations.
Fig. 7. Expression of p27 protein by EB1089 and TGF-b1. After 72 h culture of HL-60 with EB1089 (10 − 8 M), TGF-b1 (2.5 ng/ml), anti-TGF-b1 (50 mg/ml), or combinations of these agents, 100 mg of total lysates was resolved by 12% SDS polyacrylamide gel, transferred onto a nitrocellulose membrane, and probed with anti-p27 monoclonal antibody. EB1089 and TGF-b1 synergistically induced p27 protein compared to EB1089 or TGF-b1 alone. Anti-TGF-b1 partially blocked induction of p27 protein by EB1089 or TGF-b1 alone. Tubulin was used as a loading control. The p27 protein levels were quantitated using a densitometer and expressed in arbitrary units. CTR, control; EB, EB1089; T, TGF-b1; Ab, anti-TGF-b1.
eration (Fig. 2). The up-regulation of VDR mRNA was demonstrated with a peak at 4 h of exposure to TGFb1 and returned to the pretreatment level at 72 h (Fig. 6A). Western blot analysis using an anti-VDR mono-
clonal antibody showed that VDR protein expression was also up-regulated by TGF-b1 in accordance with VDR mRNA induction and the induction was apparent after 48 h of TGF-b1 treatment (Fig. 6B).
3.5. Expression of p27 by EB1089 and TGF-b1 Since cell cycle arrest by TGF-b1 is known to be mediated by p27, we examined the change of p27 protein expression in HL-60 cells at 72 h after treatment of EB1089 (10 − 8 M) and TGF-b1 (2.5 ng/ml). Both EB1089 and TGF-b1 enhanced the expression of p27 protein (Fig. 7). Signal intensity of p27 in EB1089treated HL-60 cells was stronger than TGF-b1, which was consistent in our antiproliferative data. Addition of anti-TGF-b1 to EB1089-treated HL-60 cells or TGFb1-treated HL-60 cells resulted in relatively decreased expression of p27 compared to EB1089 or TGF-b1 alone, respectively. Combined treatment of EB1089 and TGF-b1 synergistically increased the induction of p27 expression compared to either ligand alone. Fig. 6. Enhanced expression of VDR by TGF-b1. Total RNAs from HL-60 cells treated with TGF-b1 (2.5 ng/ml) for indicated times were transferred to Nytran membrane after denaturing formaldehyde gel electrophoresis and hybridized with cDNA probe of VDR. As a control, the filter was rehybridized with b-actin cDNA (A). Western blot was prepared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after cell lysis with detergent. After transfer to nitrocellulose membrane, immunoblotting was accomplished with a monoclonal anti-VDR antibody. TGF-b1 induced expression of VDR protein after 48 h of treatment. Tubulin was used as a control (B).
4. Discussion In the present study, we investigated the role of TGF-b1 in antiproliferative effect by 1,25(OH)2D3 analog, EB1089, which has more significant antitumoral activities than its parental compound, 1,25(OH)2D3. Consistent with previous study, EB1089 showed a marked growth inhibition of HL-60 cells compared to 1,25(OH)2D3 [8]. Although the precise
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antiproliferative mechanism of this vitamin D3 analog is unknown, it may be related to low affinity of vitamin D3 analog to plasma transport protein and more avid binding to its receptor [29] or to an enhanced dimerization of vitamin D receptor (VDR) with retinoic acid X receptor (RXR) compared to 1,25(OH)2D3 [30]. Acute myelogenous leukemia (AML) cells show heterogeneous response to TGF-b1 [31 – 33]. In our experiments, TGF-b1 alone had minor effect on proliferation of HL-60 cells, which is consistent with other reports [34,35]. However, combined treatment of TGF-b1 and EB1089 resulted in a significant inhibition compared to either ligand alone. The mechanism by which TGF-b1 renders HL-60 cells more sensitive to the antiproliferative activity of 1,25(OH)2D3 compound is unknown. Several lines of evidence suggest that the antiproliferative effects of several agents on malignant cells are associated with TGF-b1 [19 – 22,35]. In human osteoblasts, 1,25(OH)2D3 increased expression of TGF-b1 and TGF-b receptors [36,37]. Very recently, a positive regulation by 1,25(OH)2D3 of TGF –b1-mediated AP-1 activity was suggested in osteoblastic cells [38]. Therefore, one possible mechanism of increased antiproliferative sensitivity by EB1089 of TGF-b1 may be modulation of TGF-b1 and TGF-b1 receptor expressions. Treatment of HL-60 cells with EB1089 up-regulated the expression of TGF-b1 and TGF-b receptors, suggesting a role of TGF-b1 in antiproliferative effect of EB1089. Furthermore, to determine whether TGFb1 is involved in EB1089-induced antiproliferative effect, TGF-b1 neutralizing antibody (anti-TGF-b1) was added into HL-60 cells simultaneously treated with TGF-b1 and EB1089. Although anti-TGF-b1 can effectively neutralize endogenous TGF-b1 only under serum-free condition [39,40], we examined it under serum-containing condition, because it is difficult to culture HL-60 cells in serum-free condition, and because 1,25(OH)2D3 analogs have been shown to be more potent in the presence of serum [29]. Antiproliferative effect of EB1089 was partially blocked by antiTGF-b1. Thus, autocrine secretion of TGF-b1 may be a significant antiproliferative mechanism of EB1089 in HL-60 cells. VDR is a nuclear protein that mediates the biological actions of 1,25(OH)2D3 analogs and its level is believed to determine the cellular responsiveness [41 – 43]. In this study, we found that expression of VDR mRNA was elevated at 4 h following treatment of TGF-b1 and returned to the pretreatment level at 72 h. It is consistent with the previous reports that up-regulation of VDR mRNA in HL-60 cells by 1,25(OH)2D3 reached a peak at 1 or 2 h [41,44]. Similarly, expression of VDR protein was increased by TGF-b1, which was apparent at 48 h. Collectively, these experiments demonstrated that synergistic antiproliferative effects of EB1089 and TGF-b1 came from the enhanced expression of TGF-
b1 by EB1089 and the increased expression of VDR by TGF-b1. It has been suggested that target molecule involved in cell cycle arrest by vitamin D3 compounds is cyclin-dependent kinase inhibitor (CDKI), p21 or p27 [9,10]. However, p21 expression by 1,25(OH)2D3 was reported to be only transient and did not correlate with the onset of the G1 block in HL-60 cells [45]. Recently, Wang et al. [46] demonstrated that p27 induced G1 arrest in HL-60 cells that was reversed by an oligonucleotide antisense to p27. Asou et al. [47] reported that antiproliferative effect by a vitamin D3 analog, Ro25-9716, was associated with the induction of p27 in HL-60 cells. Therefore, it is likely that p27 is a principal mediator in vitamin D3 compound-induced cell cycle arrest. We also found that EB1089 increased the induction of p27 protein in a time- and a dose-dependent manner (data not shown). Combined treatment with TGF-b1 and EB1089 markedly increased the expression of p27 protein compared to either ligand alone, and the induction of p27 protein by TGF-b1 or EB1089 was partially blocked by anti-TGF-b1. Since p27 is known to mediate TGF-b1-induced G1 arrest [48,49], these results provide an additional evidence that TGF-b1 mediates antiproliferative effect of EB1089. In summary, EB1089 was more potent in the growth suppression of HL-60 cells than 1,25(OH)2D3. EB1089induced growth suppression was associated with the induction of TGF-b1, TGF-b RI, and TGF-b RII, and was partially reversed by the addition of anti-TGF-b1. TGF-b1 itself enhanced the expression of VDR, that explained the synergistic antiproliferative effect of TGF-b1 and EB1089. Anti-TGF-b1 partially blocked the induction of p27 by EB1089 and TGF-b1. These results suggest that the antiproliferative effect of EB1089 is mediated by TGF-b1 through p27 in HL-60 cells.
Acknowledgements This study was supported by a grant of the 1997 Korean Nation Cancer Control Program, Ministry of Health and Welfare, R.O.K. CW Jung contributed to the concept and design, drafted the manuscript, provided significant statistical expertise, administrative support and critical revision of the paper. ES Kim helped with the data collection, provided technical support, statistical assistance and interpretation of the data. JG Seol provided technical support and helped with data collection, assisted in drafting the paper and in data analysis. WH Park provided technical assistance and data collection, helped with biostatistics, analysis of data and drafting the paper. SJ Lee provided some of the funding, provided study materials and critical review of the paper. BK Kim provided some of the
C.W. Jung et al. / Leukemia Research 23 (1999) 1105–1112
funds, study materials and gave critical revision. YY Lee contributed in a major way to the study concept, gave final approval, helped with data analysis, assembly of data, administrative support, partial funding of the project and provided critical revision of the paper.
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