Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
MOLECULAR CARCINOGENESIS 37:39–50 (2003) Molecular Mechanisms of G0/G1 Cell-Cycle Arrest and Apoptosis Induced by Terfenadine in Human Cancer Cells Jean-Dean Liu,1 Ying-Jan Wang,2 Chien-Ho Chen,3 Cheng-Fei Yu,3 Li-Ching Chen,4 Jen-Kun Lin,5 Yu-Chih Liang,1 Shyr-Yi Lin,1 and Yuan-Soon Ho3* 1 Department of Internal Medicine, School of Medicine, Taipei Medical University and Hospital, Taipei, Taiwan Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan 3 Institute of Biomedical Technology, Taipei Medical University, Taipei, Taiwan 4 Department of Nursing, TzuChi University, Hualien, Taiwan 5 Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan 2 Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G0/G1 phase cell-cycle arrest. The minimal dose of TF induced significant G0 /G1 arrest in these cells was 1–3 mM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin-dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF-treated cells. On the other hand, significant apoptosis, but not G0 /G1 arrest, was induced in the HL 60 (p53-null) or Hep 3B (with deleted p53) cells when treated with TF (3–5 mM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G0 /G1 arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF-treated group was clearly attenuated by pretreatment with p27/Kip1–specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF-induced apoptosis. We also demonstrated that the TF-induced G0 /G1 cell-cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF-treated normal human fibroblast, even at a dose as high as 25 mM. Our study showed the molecular mechanisms for TF-induced cell growth inhibition and the occurrence of apoptosis in human cancer cells. ß 2003 Wiley-Liss, Inc. Key words: apoptosis; G0 /G1 cell-cycle arrest; terfenadine; p53; p21/Cip1; p27/Kip1 INTRODUCTION The occurrence of gastrointestinal (GI) cancers has increased strikingly during the last decade. For instance, colorectal cancer is the second leading cause of cancer mortality in Western societies [1] and one of the world’s most common malignancies [2,3]. Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide [4], showing the highest prevalence in Asia and Africa [5,6]; it is the second most common form of lethal cancer in China after gastric cancer [7,8]. The incidence of HCC is generally low in Western Europe and the United States. However, following an increase in hepatitis C virus infection rates, it is now occurring with increasing frequency in the United States [9]. Furthermore, HCC and colorectal cancer are the second and third causes of all cancer deaths respectively in Taiwan [10–13]. Therefore, fighting against GI cancer is an important global issue. To our knowledge, the ability of chemotherapeutic agents to inhibit cancer cell growth and to initiate apoptosis is an important ß 2003 WILEY-LISS, INC. determinant of their therapeutic response. Previously, our in vitro and in vivo studies demonstrated that antifungal agents, including miconazole, ketoconazole (KT), and griseofulvin, exert antitumor effects in various types of human GI cancers cell lines through the induction of apoptosis and cell-cycle arrest [14– 17]. In addition, our recent study also demonstrated *Correspondence to: Graduate Institute of Biomedical Technology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Received 12 September 2002; Revised 16 January 2003; Accepted 18 March 2003 Abbreviations: GI, gastrointestinal; HCC, hepatocellular carcinoma; KT, ketoconazole; TF, terfenadine; CYP 3A4, cytochrome p450-3A; ATCC, American Type Culture Collection; FCS, fetal calf serum; DMSO, dimethyl-sulfoxide; SDS-PAGE, sodium dodesyl sulfate-polyacrylamide gel electrophoresis; CDK, cyclin-dependent kinase; ODNs, oligodeoxynucleotides. DOI 10.1002/mc.10118 40 LIU ET AL. that significant apoptosis is easily induced by terfenadine (TF) in human colon and liver cancer cells [18]. TF was first reported by Kinsolving et al. in 1975 [19]. It appears to be the first highly potent H1 histamine receptor antagonist that in clinically effective doses lacks side effects, such as sedation, impaired psychomotor performance, and excessive mucosal drying [19–21]. The metabolisms of TF to its desalkyl and hydroxymetabolites are demonstrated to be mediated by human liver microsomal enzymes cytochrome p450-3A (CYP 3A4) isoforms [22,23]. KT, an antifungal drug, is a potent and selective inhibitor of the CYP 3A4 enzyme [24,25]. Our recent study demonstrated that TF-induced apoptosis is significantly potentiated by KT through the inhibition of CYP 3A4 in the HCC cell line [18]. In this study, we demonstrated for the first time that TF induced G0/ G1 cell-cycle arrest in human cancer cells, including Hep G2 (wild type p53), HT 29 (mutated p53), and COLO 205 (wild type p53) cells. In contrast, apoptosis, but not G0/G1 arrest, was induced in HL 60 (p53-null) or Hep 3B (with deleted p53) cells treated with the same dose of TF. The proteins that regulate the G0/G1 phase cell cycle and apoptosis induced by TF were determined in this study. Our results provided direct evidence that additional cytotoxic mechanisms (induction of apoptosis and cell growth inhibition) induced by TF were found in some types of human cancer cell lines. MATERIALS AND METHODS Chemicals TF was purchased from Sigma Chemical Co. (St. Louis, MO). The protein assay kit was purchased from Bio-Rad Co. (Bio-Rad Labs, Hercules, CA). Cell Lines and Cell Culture The HT 29 and COLO 205 cell lines were isolated from human colon adenocarcinoma (American Type Culture Collection [ATCC, Manassas, VA] HTB-38 and CCL-222) [26]. Hep 3B and Hep G2 cell lines were derived from human HCC (ATCC HB-8064 and HB8065) [27,28]. The HL 60 cell line was derived from human myeloid leukemia cells (ATCC 59170). The cell line CCD-922SK (ATCC CRL 1828) was derived from normal human fibroblasts. The p53 gene in the COLO 205, CCD-922SK, and Hep G2 cells was wildtype [29,30]. In contrast, the p53 gene is mutated in codon 273 in HT 29 cells [31]. The p53 gene was found to be partially deleted (7 kb) in Hep 3B [28] and null in HL 60 cells [32]. Cell lines were grown at 378C in 5% carbon dioxide atmosphere in Eagle’s minimal essential medium for CCD-922SK, Hep 3B, and Hep G2 cells, and in RPMI 1640 for COLO 205, HT 29, and HL 60 cell, supplemented with 10% fetal calf serum (FCS), 50 mg/mL gentamycin, and 0.3 mg/ mL glutamine. Determination of Cell Growth Curve Human cancer (1 104) and fibroblast (10 104) cells were plated in 35-mm Petri dishes. The next day, the medium was changed and TF (0.1–5 mM) was added. Control cells were treated with dimethylsulfoxide (DMSO) in a final concentration of 0.05% (v/v). The incubation medium was renewed every day during the experiment. At the end of incubation, cells were harvested for cell count with a hemocytometer. Cell Synchronization, Drug Treatment, and Flow Cytometry Analysis Twenty-four hours after plating of cells, the medium was removed. Cells were washed three times with medium alone, and then incubated with medium containing 0.04% FCS for 24 h. Under such conditions, cells were arrested in G0/G1, as determined by flow cytometry analysis. The low-serum medium was removed, and the cells were then stimulated by the addition of medium containing 10% FCS. TF solutions were prepared by dissolving this compound in a final concentration of 0.05% (v/v) DMSO. The stages of cell cycle in the TF- and mock-treated groups were measured by flow cytometry analysis. Western Analysis Treated and untreated cells were rinsed three times with ice-cold phosphate-buffered saline, then lysed in 500 mL of freshly prepared extraction buffer (10 mM Tris-HCl, pH 7; 140 mM sodium chloride; 3 mM magnesium chloride; 0.5% [v/v] NP-40; 2 mM phenylmethylsulfonyl fluoride; 1% [w/v] aprotinin; and 5 mM dithiothreitol) for 20 min on ice. The extracts were centrifuged for 30 min at 10 000 g. Proteins were loaded at 100 mg/lane on 12.5% [w/v] sodium dodesyl sulfate (SDS)-polyacrylamide gel, blotted, and probed with antibodies including cyclin E (Santa Cruz, Biotechnology, Inc., Santa Cruz, CA), p53, p21/CIP1, p27/Kip1, cyclin A, cyclin D1, cyclin D3, PCNA, cyclin B1, cyclin-dependent kinase 2 (CDK2), CDK4, and GAPDH (Transduction Laboratories, Lexington, KY). Immunoreactive bands were visualized by incubating with the colorigenic substrates, nitro blue tetrazolium, and 5-bromo-4chloro-3-indolyl-phosphate (Sigma). Immunoprecipitation and CDK Kinase Activity Assay CDK2-associated histone H1 kinase activity was determined as described by Wu et al. [33]. Briefly, with anti-p21/Cip1 antibody (2 mg) and protein A agarose beads (20 mL), the p21/Cip1-associated CDK2 was precipitated from 200 mg of protein lysate per sample as described above. Beads were washed three times with lysis buffer and then once with kinase assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, and 1 mM DTT). Phosphorylation of histone H1 was TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS measured by incubating the beads with 40 mL of ‘‘hot’’ kinase solution (0.25 mL [2.5 mg] of histone H1, 0.5 mL of [g-32P] ATP, 0.5 mL of 0.1 mM ATP, and 38.75 mL of kinase buffer) for 30 min at 378C. The reactions were stopped by boiling the samples in SDS sample buffer for 5 min. The samples were analyzed by 12% SDS-PAGE, and the gel was dried and subjected to autoradiography. Similarly, CDK4-Rb kinase activity was also determined as described by Wu et al. [33] with some modifications. Briefly, TF-treated cells were lysed in Rb lysis buffer (50 mM HEPES-KOH, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween-20, 10% glycerol, 80 mM b-glycerophosphate, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/mL leupetin and aprotinin), and immunoprecipited with anti-p21/Cip1 antibody (2 mg) and protein A agarose beads (20 mL). The p21/Cip1-associated CDK4 in beads were washed twice with Rb lysis buffer and then once with Rb kinase assay buffer (50 mM HEPES-KOH, pH 7.5, containing 2.5 mM EGTA, 10 mM b-glycerophosphate, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 10 mM MgCl2, and 1 mM DTT). Phosphorylation of Rb was measured by incubating the beads with 40 mL of hot Rb kinase solution (0.25 mL [2 mg] of Rb-GST fusion protein, 0.5 mL of [g-32P] ATP, 0.5 mL of 0.1 mM ATP, and 38.75 mL of Rb kinase buffer) for 30 min at 378C. The reaction was stopped by boiling the samples in SDS sample buffer for 5 min. The samples were analyzed by 12.5% SDS-PAGE, and the gel was dried and subjected to autoradiography. Determination of Apoptosis As in our previous studies, apoptosis was judged by four methods: (a) observation of the morphological changes in cells as described previously [30]; (b) translocation of phosphotidyl serine to the cell surface detected by an Annexin V-FITC apoptosis detection kit (Calbiochem, Bad Soden, Germany) [34]; (c) the presence of sub-G1 peak detected by flow cytometry on a FACSCalibur (Becton Dickinson, Heidelberg, Germany) [35]; and (d) the appearance of DNA fragmentation analyzed by the methods described previously [16]. Antisense Oligodeoxynucleotide (ODN) Transfection Procedures The p27/Kip1-specific antisense ODN sequence used in the experiments was (50 -TCTTCTGTTCTGTTGGCCCT-30 ); and sense ODN sequence was (50 CGTGAGAGTGTCTAACGGGAG-30 ) [36]. The p21/ Cip1-specific antisense ODNs (50 -UCCGCGCCCAGCUCC-30 ); and sense ODNs (50 -UCCGCCCGCAGUCCC-30 ) [37] phosphothioates (S-oligos) were synthesized and purified with high-performance liquid chromatography by Genset. Antisense or 41 sense ODNs were added to the HL 60 cells at a final concentration of 20 mM 16 h before the cells were challenged with 10% FCS and TF (10 mM) treatment for additional 15 h. The methods for transfection of the ODNs were the same as in our previous report [14]. RESULTS TF Induces G0 /G1 Phase Cell-Cycle Arrest in Human Cancer Cells In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells including hepatoma (Hep G2 and Hep 3B), colon cancer (HT 29 and COLO 205), and leukemia (HL 60) cells (Figure 1A and E). Furthermore, similar results were also observed in the human normal fibroblast treated with higher doses of TF (1–5 mM) (Figure 1F), indicating that TF-induced cell growth inhibition and cytotoxic effects were dose- and cell line– dependent. In order to determine whether TF could induce cell-cycle arrest in human cells, HT 29 and COLO 205 cells were synchronized at the G0/G1 phase by 0.04% serum starvation for 24 h and then treated with complete medium (with 10% FCS) containing TF (5 mM). Flow cytometry analysis was determined at the indicative time points. Our results showed that TF (5 mM) treatment caused an apparent G0/G1 phase arrest in COLO 205 (85.3%) and HT 29 cells (more than 90%) (Figure 2D and H). Normal cell-cycle progression was observed in DMSO-treated cells after being challenged with complete medium without TF. Figure 2D and H demonstrates that the most significant difference of the G0/G1 phase cell population between the TF- and DMSO-treated groups is at 15 h after restimulation with complete medium. Accordingly, this time point (15 h) was selected for studying the dose-dependent effect of TF-induced G0/G1 arrest in various types of human cancer and fibroblast cells (Figure 3A–F). Our results revealed that G0/G1 arrest was induced significantly in the COLO 205 and the Hep G2 cells treated with TF (1–3 mM). However, apoptosis occurred when these two types of cells were treated with a higher dose of TF (>15 mM) (Figure 3B and C). Interestingly, G0/G1 cell-cycle arrest, but not apoptosis, was observed in the HT 29 cells, even with a higher dose of TF (30 mM). In contrast, apoptosis was more significantly induced in the Hep 3B and the HL 60 cells treated with TF (3 mM) (Figure 3D and E). Importantly, neither apoptosis nor cell growth cycle arrest was observed in human normal fibroblast, even with a higher dose of TF (25 mM) (Figure 3F). From these data, we found that induction of the G0/G1 phase cell-cycle arrest and apoptosis were observed in the p53-expression (COLO 205, Hep G2, and HT 29) cells, while the occurrence of apoptosis was more easily induced in the p53-deficient (Hep 3B and HL 60) cells when 42 LIU ET AL. Figure 1. Viability of human cancer and fibroblast cells treated with TF. COLO 205 (A), HT 29 (B), Hep G2 (C), Hep 3B (D), HL 60 (E), and fibroblast (CCD 922SK) (F) cells were treated with TF (0.1–5 mM) at the indicated time points. The viability was then determined by trypan blue exclusion assay as described in Materials and Methods. Results were the mean of three independent experiments. exposed to TF. The TF-induced G0/G1 cell-cycle arrest was not reversed by TF removal, and this growth inhibition lasted for at least 7 d (data not shown). TF-Induced Apoptosis in Various Types of Human Cancer Cell Lines Our recent study [35] demonstrated that apoptosis can be induced in Hep G2 and COLO 205 cells that have suffered a higher dose of TF (>10 mM) treatment. In this study, the cells treated with TF (15 mM) exhibited morphological changes (data not shown) as well as progressive internucleosomal degradation of DNA, yielding a ladder of DNA fragments (Figure 4). Interestingly, at such a high dose (15 mM) of TF, apoptosis was more easily induced in the p53-null (HL 60 and Hep 3B) cells compared to the p53 wild type (Hep G2 and COLO 205) cells (Figures 3 and 4). Our data demonstrated that either apoptosis or G0/ G1 cell-cycle arrest effects induced by TF depended on TF dosage and cell type. Molecular Mechanisms of TF-Induced G0/G1 Arrest As shown in Figure 3, significant G0/G1 arrest was induced by TF (>5 mM) in both HT 29 and COLO 205 cells. COLO 205 (with wild type p53) cells were then selected to investigate the molecular mechanisms of TF-induced G0/G1 cell-cycle arrest. The COLO 205 cells were synchronized at the G0/G1 phase as described previously [17]. The cells were then exposed to 5 mM TF and immunoblotting analysis was performed time-dependently. The time points that we selected for drug treatment were, as in our previous paper [17], reported as: 0 h (representing the G0/G1 phase), 15 h (representing the S phase), 18 h (representing the G2/M phase), and 24 h (representing the second G0/G1 phase). The expression of p21/ Cip1, a key regulator of cell entry into mitosis, was monitored by immunoblotting analysis. Our results revealed that the p21/Cip1 protein was induced initially at 15 h and persisted for at least 24 h by TF TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS 43 Figure 2. Time-dependent response of TF-induced G0 /G1 phase arrest in COLO 205 and HT 29 cells. COLO 205 (A–D), and HT 29 (E– H) cells were synchronized with 0.04% FCS for 24 h as described in Materials and Methods. After synchronization, cells were then released into complete medium (10% FCS) containing DMSO (0.05%), and TF (5 mM). The DNA content distribution histograms of FACS analysis were measured by flow cytometry at the indicated time points (0 and 18 h) after TF treatment. Different phases of the cell cycle were determined with established CellFIT DNA analysis software. Each blot is representative of three similar experiments. Significance was accepted at **P < 0.01. treatment (Figure 5). Our results also showed that the level of cyclin D1, cyclin D3, cyclin E, PCNA, CDK2, and CDK4 in TF-treated cells was not significantly changed at 15 h by TF-treatment (Figure 5). p27/Kip1, which inhibits CDK2 kinase activity, was also analyzed for its expression level. Our results indicated that the p27/Kip1 protein level was significantly elevated at 15 h after TF-treatment. In contrast, cyclin A and cyclin B, which promote cell entry from G0/G1 into S and from S into the G2/M phase, respectively, were downregulated in TFtreated cells (Figure 5). Previous studies demon- strated that the p53 protein is a potent transcription factor, activated and accumulated in response to DNA-damaging agents [38–40], leading to cell-cycle arrest, or apoptosis [41,42]. Our results in Figures 2B and 5 demonstrated that G0/G1 cell-cycle arrest and induction of the p53 protein expression were observed concomitantly in COLO 205 cells after 5 mM TF treatment. These findings suggested that the p53signaling pathway was involved in the regulation of TF-induced G0/G1 cell-cycle arrest. To further scrutinize such observations, human colon cancer cell lines with wild-type (COLO 205) or 44 LIU ET AL. Figure 3. TF-induced G0 /G1 phase cell-cycle arrest and apoptosis in human cancer cells. HT 29 (A), COLO 205 (B), Hep G2 (C), Hep 3B (D), HL 60 (E), and human fibroblast (CCD 922SK) (F) cells were treated by TF dose-dependently. FACS analysis of DNA content was performed at 15 h after release from quiescence by incubation in culture media supplemented with 10% FCS and various concentrations of TF (0.1–30 mM) in 0.05% DMSO. Percentages of cells in the Sub-G1, G0 /G1, S, and G2/M phases of the cell cycle were determined with established CellFIT DNA analysis software. Three samples were analyzed in each group, and values represent the mean SE. mutated p53 (HT 29) status, and human leukemia cell line (HL 60) with nulled p53 were selected to verify the role of p53 in response to TF treatment. All cells within each category were treated with TF (0.1–10 mM) dose-dependently for 15 h, then the cells were harvested and subjected to Western blot analysis with antibodies as illustrated in Figure 6. The p53 and p21/Cip1 protein levels were elevated significantly in the HT 29 and COLO 205 cells (Figure 6). Interestingly, the cyclin D3 and CDK4 protein expression in all these cells were downregulated dose-dependently. As shown in Figure 6, induction TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS Figure 4. DNA fragmentation analysis in human cancer cells undergoing TF-induced apoptosis. Human cancer cells, including HT 29 (lane 1), COLO 205 (lane 2), Hep G2 (lane 3), HL 60 (lane 4), Hep 3B (lane 5), and normal fibroblast (lane 6) cells were treated with TF (15 mM). DNA fragmentation analysis was examined 24-h later (upper panel). Cells in the lower panel received mock treatment as controls. of p21/Cip1 was only observed in the HL 60 cells treated with a higher dose of TF (>10 mM). Importantly, at such a dose of TF (>10 mM), significant apoptosis instead of G0/G1 arrest was observed in HL 60 cells (Figure 3E). As shown in Figure 7, protein levels of p53, p27/ Kip1, CDK2-associated p21/Cip1, and CDK4-associated p21/Cip1 were induced in the TF (5 mM)treated HT 29 and COLO 205 cells (Figure 7A). Kinase assays revealed that the activities of CDK2 and CDK4 in the TF-treated COLO 205 and HT 29 cells were inhibited (Figure 7B). As described previously, p21/ Cip1 was a potent inhibitor of CDK2/4. Our data revealed that CDK2 and CDK4 kinase activities were inhibited concomitantly with the increased binding of p21/Cip1 to CDK2 and CDK4 in cells treated with TF (Figure 7). Collectively, our data indicated that G0/G1 arrest induced by TF was due to decreased activity of CDKs mediated by an increase of p21/ 45 Figure 5. Time-dependent effect of TF on cell-cycle regulatory protein levels in COLO 205 cells. The COLO 205 cells were synchronized with 0.04% FCS for 24 h as described in Materials and Methods. After synchronization, cells were then released into complete medium (10% FCS) containing TF (5 mM) and DMSO (0.05%, v/v) for the indicated time points. Protein extracts (100 mg/ lane) were separated by SDS-PAGE, probed with specific antibodies, and detected with the NBT/BCIP system. Cip1-CDKs association. We further investigated the phosphorylation status of the pRb and the association of pRb with E2F in response to TF. Figure 7A shows that pRb was remarkably dephosphorylated at 15 h after TF treatment, and the hypophosphorylated pRb-E2F complexes were increased at 15 h after TF treatment in the COLO 205 and HT 29 cells. In contrast, the levels of the pRb hypophosphorylation and the pRb-E2F complexes were not changed significantly in the TF-treated HL 60 cells. As shown in Figure 3D and E, apoptotic cells were detected in the Hep 3B and HL 60 cells treated with a lower dose of TF (3–5 mM). Such effects implied that some regulatory proteins were involved in apoptosis induction in response to TF treatment. Previous studies indicated that the p27/Kip1 protein is activated during the apoptosis process induced by different agents [43–47]. The p27/Kip1 expression was easily induced by TF treatment in both of the HL 60 46 LIU ET AL. Figure 6. Dose-dependent effect of TF on the cell-cycle regulatory protein levels in human cancer cells. Human cancer cells with different p53 status, including COLO 205 (wild type p53), HT 29 (mutated p53), and HL 60 (p53-null) cells were rendered quiescent by incubation for 24 h in the cultured media containing 0.04% FCS. Cells were then challenged with 10% FCS and treated with various concentrations of TF (0.1–10 mM) for additional 15 h. Protein extracts (100 mg/lane) were separated by SDS-PAGE, probed with specific antibodies, and detected with the NBT/BCIP system. Membranes were also probed with anti-GAPDH antibody to correct for differences in protein loading. and COLO 205 cells (Figures 6 and 7). In contrast, significant induction of the p27/Kip1 was detected in HT 29 cells treated with a higher dose of TF (>10 mM) (Figure 6). Such results implied that p27/Kip1 might play an important role in the TF-induced apoptosis observed in the COLO 205 and HL 60 cells. protein expression levels were monitored by immunoblotting analysis (Figure 8). Figure 3E demonstrated that apoptosis was induced by TF in the HL 60 cells dose-dependently. HL 60 cells were selected for further clarifying the roles of p27/Kip1 involved in TF-induced apoptosis. The HL 60 cells were treated with 20 mM of p27/Kip1-specific antisense ODNs for 16 h and then exposed to TF (15 mM) for another 15 h. Our results revealed that the TF-induced p27/Kip1 protein expression was attenuated by the p27/Kip1specific antisense ODNs (Figure 8A, lane 3), but not by its sense ODNs (Figure 8A, lane 5). To examine further whether the TF-induced apoptosis would be reduced by the p27/Kip1-specific antisense ODNs, the apoptotic cells were detected by annexin V/PI analysis. Our data demonstrated that the percentage of TF-induced apoptotic-cell population clearly decreased when pretreated with the p27/Kip1-specific ODNs (Figure 8A, bar 3). To confirm such observations, similar experiments were performed and the p27/Kip1-(Figure 8B) or p21/ Cip1-specific (Figure 8C) antisense ODNs (20 mM) were added to COLO 205 cells for 16 h and then exposed to TF (15 mM) for another 15 h. Our results p27/Kip1, Not p21/Cip1, Plays a Major Role in TF-Induced Apoptosis in Human HL 60 and COLO 205 Cells As described previously, p21/Cip1 and p27/kip1 were demonstrated as negative regulators of cyclin and CDK activity and appear to be both essential and sufficient to arrest cells before the late G1 restriction point [48,49]. However, previous study demonstrated that adenovirus-mediated p27/Kip1 overexpression leads to apoptosis in human cancer cells. In sharp contrast, a similar overexpression of p21/Cip1 results in G1-S arrest but minimum cytotoxicity [50]. To clarify the roles of the p21/ Cip1 and the p27/Kip1 protein expression that was involved in G0/G1 arrest and apoptosis induced by TF in human cancer cells, antisense ODNs specific to the p21/Cip1 and the p27/Kip1 were used, and the TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS 47 Figure 7. The G0 /G1 phase regulatory proteins were involved in TF-induced human cancer cells growth inhibition. (A) Human cancer cells with different p53 status, including HT 29 (p53 mutant), COLO 205 (p53 wild type), and HL 60 (p53-null) cells, were treated with 5 mM TF (þ) or 0.05% (v/v) DMSO () for 15 h after release from quiescence. Protein extracts (100 mg/lane) were separated by SDSPAGE, probed with specific antibodies, and detected with the NBT/ BCIP system. (B) CDK2 and CDK4 kinase activity were determined as described in Materials and Methods. revealed that TF-induced apoptosis in the COLO 205 cells was significantly attenuated by the p27/Kip1specific antisense ODNs (Figure 8B, lane 3, and bar 3). In contrast, the TF-induced apoptosis in the COLO 205 cells was not inhibited by the p21/Cip1-specific ODNs (Figure 8C, lane 3, and bar 3). Our results demonstrated that the p27/Kip1 protein might play an important role in the TF-induced apoptosis in both COLO 205 and HL 60 cells. DISCUSSION TF-Induced Human Cancer Cell-Cycle Arrest Was Through the p53-Dependent and p53-Independent Signaling Pathways The p53 tumor suppressor is a predominantly nuclear transcription factor, activated by various stresses including chemotherapeutic and chemopreventive agents [51]. Although analysis of a large number of human tumor-derived cell lines has suggested a link between p53-status and drug- Figure 8. p27/Kip1 protein expression plays a critical role in TFinduced apoptosis in COLO 205 and HL 60 cells. Antisense or sense ODNs specific to p27/Kip1 and p21/Cip1 were added to HL 60 (A) and COLO 205 (B and C) cells at a final concentration of 20 mM at 16 h before the cell was challenged with 10% FCS and 15 mM TF treatment for additional 15 h. The levels of p27/Kip1, p21/Cip1, and GAPDH proteins were determined by Western blot analysis. Percentage of apoptotic cells was determined by with annexin V/PI analysis shown in the bottom chart. M, size marker; AS, antisense oligonucleotide; S, sense oligonucleotide. sensitivity [52], the role of p53 involved in the response of human tumor cells to chemotherapeutic agents is controversial [53]. In this study, human cancer cells (Hep G2 and COLO 205) with wild-type p53 status were more sensitive to TF-induced G0/G1 cell-cycle arrest. Our recent studies demonstrated that G0/G1 cell-cycle arrest is induced by antifungal agents (such as KT and miconazole) in the COLO 205 48 LIU ET AL. cells [14,17]. All of these agents induce p53, p21/ Cip1, and p27/Kip1 protein expression in the COLO 205 cells via a p53-dependent signaling pathway [14,17]. In this study, we further demonstrated that significant G0/G1 cell-cycle arrest was also induced by TF in HT 29 (p53 His273 mutant) cells (Figure 2H). We demonstrated that p21/Cip1 protein was markedly induced in p53-mutated (HT 29) cells (Figure 6). Such results implied that p21/Cip1 protein expression in HT 29 cells was induced by TF in a p53independent signaling pathway. Our observations were similar to those of previous studies that indicated that p21/Cip1 protein expression can be stimulated by a variety of transcriptional activators other than p53 that are often associated with growth arrest and differentiation (such as MyoD7, STAT18, and BRCA19) [54–56]. Direct evidence of p53regulated TF-induced p21/Cip1 protein expression and its association with G0/G1 arrest will be investigated in our further study by the p53-specific antisense ODNs as described recently [14]. TF-Induced Apoptosis Tended to Occur in HL 60 and Hep 3B Cells Our recent report [35] and this study (Figures 3B and C) demonstrated that apoptosis can be induced in Hep G2 and COLO 205 cells with a higher dose of TF (>10 mM). Importantly, when the COLO 205 and Hep G2 cells were exposed to TF at a lower dose (0.1– 5 mM), G0/G1 cell-cycle arrest instead of apoptosis was induced within 24 h. To avoid the occurrence of apoptosis and specifically investigate the TF-induced G0/G1 cell-cycle arrest in COLO 205 and Hep G2 cells, the dose of TF was limited to a narrow range of 0.1–5 mM. Moreover, we also demonstrated that apoptosis tended to be induced by TF in the p53-null cells (HL 60 and Hep 3B) at this dosage range. Our data demonstrated that either apoptosis or G0/G1 cell-cycle arrest induced by TF was a dose- and cell type–dependent effect. Such results implied that the p53-signaling pathway might play an important role in G0/G1 cell-cycle arrest as a response to TF treatment instead of apoptosis. To our knowledge, the p21/Cip1 is a negative regulator of cell-cycle check point and is a transcriptional target of p53 [57,58]. However, previous studies have established a role for p21/Cip1 as a survival factor by demonstrating that defective p21/ Cip1 expression can lead to human colon cancer cells’ (HCT116) undergoing apoptosis instead of cellcycle arrest [59,60]. In this study, significant G0/G1 phase cell-cycle arrest instead of apoptosis was found in HT 29 cells after a higher dose of TF (30 mM). Our results suggest that p21/Cip1 protein induction might play a key regulatory role in TF-induced protection of apoptosis in HT 29 cells. Additional experiments may be needed to clarify the role of p21/Cip1 protein expression involved in TF-induced G0/G1 arrest in human HT 29 cells. p27/Kip1 Protein Was the Key Regulator in TF-Induced Apoptosis in Human Cancer Cells As described above, apoptosis was significantly induced by a lower dose of TF (3–5 mM) in HL 60 cells. However, p21/Cip1 protein induction in the HL 60 cells was only observed at a higher dose of TF (>10 mM). Such results suggested that p21/Cip1 protein was not playing a role in the prevention of TF-induced apoptosis in HL 60 cells. Additional regulatory proteins may be involved in TF-induced apoptosis. Previous studies demonstrated that p27/ Kip1 protein plays an important role in human cancer cell apoptosis induced by various stimuli [61– 64]. To further scrutinize the role of p21/Cip1 and p27/Kip1 involved in TF-induced apoptosis, antisense ODNs specific to the p21/Cip1 and p27/Kip1 were added in cultured HL 60 cells treated with higher dose of TF (10 mM). The induction of apoptosis is significantly attenuated by the p27/Kip1-specific ODNs, whereas the p21/Cip1-specific ODNs are not. Such observations demonstrated that p27/Kip1 is involved in TF-induced apoptosis in HL 60 cells. As described previously [59,60,65], p21/Cip1 induction is involved in the prevention of apoptosis in human colon carcinoma cells. However, our study demonstrated that induction of p21/Cip1 was not involved in TF-induced apoptosis in HL 60 cells, as evidenced by the antisense ODNs experiments (data not shown). The role of increased p21/Cip1 expression in response to TF-induced G0/G1 cell-cycle arrest and apoptosis in HT 29 and HL 60 cells might be cell type–specific and needs to be further clarified. TF-Induced G0 /G1 Arrest Was Irreversible As shown in Figure 3A, G0/G1 arrest but not apoptosis was observed in HT 29 cells in response to a higher dose (30 mM) of TF. Such results implied that HT 29 cells could be used as a candidate to illustrate the molecular mechanisms of TF-induced G0/G1 cell-cycle arrest. In this study, we demonstrated that TF-induced G0/G1 arrest was irreversible in the HT 29 cells. The antiproliferation effect of many anticancer drugs used clinically was reversible. Therefore, once the drug treatment was terminated, the growth of the tumor cell rebounded. Lately, scientists and clinicians have been searching for some new drugs that will exert an irreversible antiproliferative effect. Moreover, a drug with an irreversible effect can be applied in a lower dose for a longer period of time to achieve the same therapeutic effect as given by a higher dose of another drug, but with fewer side effects. Accordingly, we attempted to test the irreversibility of the antiproliferative effect of TF on tumor cell growth. More experiments must be performed in our further study to support this hypothesis. Our results showed the molecular basis of TF-induced cancer cell growth inhibition in vitro, and further animal experiments will be important TERFENADINE–INDUCED G0 /G1 CELL-CYCLE ARREST IN CANCER CELLS to demonstrate the potential anticancer effect of TF in vivo. ACKNOWLEDGMENTS This study was supported by the National Science Council grant NSC 90-2320-B-038-033, NSC 90-2320-B-006-086, and by the Jin Lung Yen Foundation. REFERENCES 1. Kang SK, Burnett CA, Freund E, Walker J, Lalich N, Sestito J. Gastrointestinal cancer mortality of workers in occupations with high asbestos exposures. Am J Ind Med 1997;31:713– 718. 2. Strickland L, Letson GD, Muro-Cacho CA. Gastrointestinal stromal tumors. Cancer Control 2001;8:252–261. 3. Hirota S. Gastrointestinal stromal tumors: Their origin and cause. Int J Clin Oncol 2001;6:1–5. 4. Kiyosawa K, Sodeyama T. Global epidemiology of hepatocellular carcinoma. Nippon Rinsho 2001;59:13–19. 5. Teo EK, Fock KM. Hepatocellular carcinoma: An asian perspective. Dig Dis 2001;19:263–268. 6. Kew MC. Hepatitis C virus infection in black patients with hepatocellular carcinoma in southern Africa. Princess Takamatsu Symp 1995;25:33–40. 7. London WT, Evans AA, McGlynn K, et al. Viral, host and environmental risk factors for hepatocellular carcinoma: A prospective study in Haimen City, China. Intervirology 1995; 38:155–161. 8. London WT, Evans AA, Buetow K. et al. Molecular and genetic epidemiology of hepatocellular carcinoma: Studies in China and Senegal. Princess Takamatsu Symp 1995;25: 51–60. 9. El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999;340: 745–750. 10. Lu SN, Lee CM, Changchien CS, Chen CJ. Excess mortality from hepatocellular carcinoma in an HCV-endemic township of an HBV-endemic country (Taiwan). Trans R Soc Trop Med Hyg 1999;93:600–602. 11. Chen CH, Chen DS. Hepatocellular carcinoma: 30 years’ experience in Taiwan. J Formos Med Assoc 1992;91:S187– S202. 12. Hwang WS, Yao JC, Cheng SS, Tseng HH. Primary colorectal lymphoma in Taiwan. Cancer 1992;70:575–580. 13. Chen LK, Hwang SJ, Li AF, Lin JK, Wu TC. Colorectal cancer in patients 20 years old or less in Taiwan. South Med J 2001; 94:1202–1205. 14. Chen RJ, Lee WS, Liang YC, et al. Ketoconazole induces G0/ G1 arrest in human colorectal and hepatocellular carcinoma cell lines. Toxicol Appl Pharmacol 2000;169:132–141. 15. Ho YS, Duh JS, Jeng JH, et al. Griseofulvin potentiates antitumorigenesis effects of nocodazole through induction of apoptosis and G2/M cell cycle arrest in human colorectal cancer cells. Int J Cancer 2001;91:393–401. 16. Ho YS, Tsai PW, Yu CF, Liu HL, Chen RJ, Lin JK. Ketoconazoleinduced apoptosis through p53-dependent pathway in human colorectal and hepatocellular carcinoma cell lines. Toxicol Appl Pharmacol 1998;153:39–47. 17. Wu CH, Jeng JH, Wang YJ, et al. Antitumor effects of miconazole on human colon carcinoma xenografts in nude mice through induction of apoptosis and G0/G1 cell cycle arrest. Toxicol Appl Pharmacol 2002;180:22–35. 18. Wang YJ YC, Chen LC, et al. Ketoconazole potentiates terfenadine-induced apoptosis in human Hep G2 cells through inhibition of cytochrome p450 3A4 activity. J Cell Biochem 2002;87:147–159. 19. Kinsolving CR, Munro NL. The objective and timing of drug disposition studies. Appendix V: A comparison of the bio- 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 49 availability of three dosage forms of terfenadine. Drug Metab Rev 1975;4:285–290. Kaliner MA, Check WA. Non-sedating antihistamines. Allergy Proc 1988;9:649–663. Rafferty P, Holgate ST. Terfenadine (Seldane) is a potent and selective histamine H1 receptor antagonist in asthmatic airways. Am Rev Respir Dis 1987;135:181–184. Ling KH, Leeson GA, Burmaster SD, Hook RH, Reith MK, Cheng LK. Metabolism of terfenadine associated with CYP3A(4) activity in human hepatic microsomes. Drug Metab Dispos 1995;23:631–636. Rodrigues AD, Mulford DJ, Lee RD, et al. In vitro metabolism of terfenadine by a purified recombinant fusion protein containing cytochrome P4503A4 and NADPH-P450 reductase. Comparison to human liver microsomes and precisioncut liver tissue slices. Drug Metab Dispos 1995;23:765– 775. Parker RS, Sontag TJ, Swanson JE. Cytochrome P4503Adependent metabolism of tocopherols and inhibition by sesamin. Biochem Biophys Res Commun 2000;277:531– 534. Boxenbaum H. Cytochrome P450 3A4 in vivo ketoconazole competitive inhibition: Determination of Ki and dangers associated with high clearance drugs in general. J Pharm Pharmaceutical Sci 1999;2:47–52. Semple TU, Quinn LA, Woods LK, Moore GE. Tumor and lymphoid cell lines from a patient with carcinoma of the colon for a cytotoxicity model. Cancer Res 1978;38:1345– 1355. Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980;209:497–499. Darlington GJ, Kelly JH, Buffone GJ. Growth and hepatospecific gene expression of human hepatoma cells in a defined medium. In Vitro Cell Dev Biol 1987;23:349–354. Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M. Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc Natl Acad Sci USA 1990;87:1973–1977. Ho YS, Wang YJ, Lin JK. Induction of p53 and p21/WAF1/ CIP1 expression by nitric oxide and their association with apoptosis in human cancer cells. Mol Carcinogen 1996;16: 20–31. Niewolik D, Vojtesek B, Kovarik J. p53 derived from human tumour cell lines and containing distinct point mutations can be activated to bind its consensus target sequence. Oncogene 1995;10:881–890. Ju JF, Banerjee D, Lenz HJ, et al. Restoration of wild-type p53 activity in p53-null HL-60 cells confers multidrug sensitivity. Clin Cancer Res 1998;4:1315–1322. Wu X, Rubin M, Fan Z, et al. Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factor receptor monoclonal antibody. Oncogene 1996;12:1397–1403. Tseng CJ, Wang YJ, Liang YC, et al. Microtubule damaging agents induce apoptosis in HL 60 cells and G2/M cell cycle arrest in HT 29 cells. Toxicology 2002;175:123–142. Wang YJ, Yu CF, Chen LC, et al. Ketoconazole potentiates terfenadine-induced apoptosis in human Hep G2 cells through inhibition of cytochrome p450 3A4 activity. J Cell Biochem 2002;87:147–159. Braun-Dullaeus RC, Mann MJ, Ziegler A, von der Leyen HE, Dzau VJ. A novel role for the cyclin-dependent kinase inhibitor p27(Kip1) in angiotensin II-stimulated vascular smooth muscle cell hypertrophy. J Clin Invest 1999;104: 815–823. Liu J, Estes ML, Drazba JA, et al. Anti-sense oligonucleotide of p21(waf1/cip1) prevents interleukin 4-mediated elevation of p27(kip1) in low grade astrocytoma cells. Oncogene 2000; 19:661–669. Morgan SE, Kastan MB. p53 and ATM: Cell cycle, cell death, and cancer. Adv Cancer Res 1997;71:1–25. 50 LIU ET AL. 39. Kern SE, Kinzler KW, Bruskin A, et al. Identification of p53 as a sequence-specific DNA-binding protein. Science 1991; 252:1708–1711. 40. Haffner R, Oren M. Biochemical properties and biological effects of p53. Curr Opin Genet Dev 1995;5:84–90. 41. Ko LJ, Prives C. p53: Puzzle and paradigm. Genes Dev 1996;10:1054–1072. 42. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323–331. 43. Turturro F, Frist AY, Arnold MD, Pal A, Cook GA, Seth P. Comparison of the effects of recombinant adenovirusmediated expression of wild-type p53 and p27Kip1 on cell cycle and apoptosis in SUDHL-1 cells derived from anaplastic large cell lymphoma. Leukemia 2001;15:1225–1231. 44. Donjerkovic D, Mueller CM, Scott DW. Steroid- and retinoidmediated growth arrest and apoptosis in WEHI-231 cells: Role of NF-kappaB, c-Myc and CKI p27(Kip1). Eur J Immunol 2000;30:1154–1161. 45. Choi SH, Kim SW, Choi DH, Min BH, Chun BG. Polyaminedepletion induces p27Kip1 and enhances dexamethasoneinduced G1 arrest and apoptosis in human T lymphoblastic leukemia cells. Leuk Res 2000;24:119–127. 46. Donjerkovic D, Zhang L, Scott DW. Regulation of p27Kip1 accumulation in murine B-lymphoma cells: Role of c-Myc and calcium. Cell Growth Differ 1999;10:695–704. 47. Wang X, Gorospe M, Huang Y, Holbrook NJ. p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene 1997;15:2991–2997. 48. Polyak K, Kato JY, Solomon MJ, et al. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev 1994;8:9–22. 49. Toyoshima H. Control of cell cycle by the p27 Cdk-inhibitor. Tanpakushitsu Kakusan Koso 1996;41:1732–1736. 50. Katayose Y, Kim M, Rakkar AN, Li Z, Cowan KH, Seth P. Promoting apoptosis: A novel activity associated with the cyclin-dependent kinase inhibitor p27. Cancer Res 1997;57: 5441–5445. 51. Jimenez GS, Khan SH, Stommel JM, Wahl GM. p53 regulation by post-translational modification and nuclear retention in response to diverse stresses. Oncogene 1999; 18:7656–7665. 52. Brown JM, Wouters BG. Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res 1999;59:1391– 1399. 53. Weinstein JN, Myers TG, O’Connor PM, et al. An information-intensive approach to the molecular pharmacology of cancer. Science 1997;275:343–349. 54. Halevy O, Novitch BG, Spicer DB, et al. Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science 1995;267:1018–1021. 55. Chin YE, Kitagawa M, Su WC, You ZH, Iwamoto Y, Fu XY. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1. Science 1996; 272:719–722. 56. Somasundaram K, Zhang H, Zeng YX, et al. Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDKinhibitor p21WAF1/CiP1. Nature 1997;389:187–190. 57. Waldman T, Kinzler KW, Vogelstein B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res 1995;55:5187–5190. 58. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998;282:1497–1501. 59. Bunz F, Hwang PM, Torrance C, et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest 1999;104:263–269. 60. Waldman T, Lengauer C, Kinzler KW, Vogelstein B. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 1996;381:713–716. 61. Huang L, Pardee AB. Beta-lapachone induces cell cycle arrest and apoptosis in human colon cancer cells. Mol Med 1999;5:711–720. 62. Gil-Gomez G, Berns A, Brady HJ. A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis. EMBO J 1998;17:7209–7218. 63. Fujieda S, Inuzuka M, Tanaka N, et al. Expression of p27 is associated with Bax expression and spontaneous apoptosis in oral and oropharyngeal carcinoma. Int J Cancer 1999;84: 315–320. 64. Hayashi K, Yokozaki H, Naka K, Yasui W, Lotan R, Tahara E. Overexpression of retinoic acid receptor beta induces growth arrest and apoptosis in oral cancer cell lines. Jpn J Cancer Res 2001;92:42–50. 65. Mahyar-Roemer M, Roemer K. p21 Waf1/Cip1 can protect human colon carcinoma cells against p53-dependent and p53-independent apoptosis induced by natural chemopreventive and therapeutic agents. Oncogene 2001;20:3387– 3398.