261 Journal of Oral Science, Vol. 43, No. 4, 261-268, 2001 Comparative study of calcium-channel blockers on cell proliferation, DNA and collagen syntheses, and EGF receptors of cultured gingival fibroblasts derived from human nifedipine, nicardipine and nisoldipine responders Hiroko Matsumoto•˜, Ichinari Noji•˜, Yoshiaki Akimoto•õ and Akira Fujii•˜ §Department of Pharmacology and †Second Department of Oral Surgery, Nihon University School of Dentistry at Matsudo, Chiba 271-8587 (Received 6 September and accepted 4 December 2001) Abstract: Our previous study indicated that Key words: calcium-channel blockers; gingival fibroblasts derived from patients reactive to nifedipine overgrowth; human gingival fibroblasts might be susceptible to the other calcium-channel (HGF). blockers (nicardipine, verapamil and diltiazem) in terms of cell proliferation, DNA synthesis, and collagen synthesis. Thus, the present investigation was designed Introduction to clarify the cross-reactivity among dihydropyridine Many case reports have implicated nifedipine, one of calcium-channel blockers (nifedipine, nicardipine, and the dihydropyridine calcium-channel blockers, as a cause nisoldipine). Human gingival fibroblasts derived from of gingival overgrowth (first reported by Ramon et al. (1) seven, two, and one patients who developed gingival and Lederman et al. (2)). The incidence of gingival overgrowth as a result of nifedipine, nicardipine, and overgrowth due to nifedipine has been reported to be 6.5% nisoldipine medications, respectively, were examined (3), more than 10% (4), 13.7% (5), 15% (6), and 20% (7). in terms of the effect of calcium-channel blockers This unwanted side effect has also been reported in patients (nifedipine, diltiazem, verapamil, and nicardipine) on taking three other calcium-channel blockers; diltiazem cell proliferation, DNA synthesis, collagen synthesis, (8-10), verapamil (11-13), and nicardipine (5,14). We and the number of epidermal growth factor (EGF) have recently found a patient who developed gingival receptors. Phenytoin was used as a positive control. With overgrowth as a result of nisoldipine medication. Excellent most of the calcium-channel blockers and phenytoin, reviews on drug-induced gingival overgrowth are available fibroblasts from patients reactive to nifedipine and (15-17). Nifedipine is increasingly used in the treatment nicardipine medications gave a better cell proliferation of angina pectoris and hypertension and is the most rate, DNA synthesis, and an increased number of EGF frequently cited causal agent in calcium-channel blocker- receptors, compared to non-drug-treated control. induced gingival overgrowth (4). However, this was not the case for calcium-channel We have previously demonstrated that the fibroblasts blockers tested in fibroblasts from patients reactive to from patients reactive to nifedipine gave trends toward better nisoldipine medication. (J. Oral Sci. 43, 261-268, 2001) cell proliferation rates, DNA synthesis, and collagen synthesis than those from non-reactive patients in the Correspondence to Dr. Akira Fujii, Department of presence of 1 ƒÊM of calcium-channel blockers (nifedipine, Pharmacology, Nihon University School of Dentistry at diltiazem, nicardipine, and verapamil) or phenytoin (17). Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Therefore, it is possible that fibroblasts from reactive Japan patients may be also susceptible to the other calcium- Tel: +81-47-360-9344 channel blockers, which indicates that those patients who Fax: +81-47-360-9348 E-mail address: [email protected] developed gingival overgrowth because of nifedipine 262 medication may also develop it in response to other Ltd., Japan). The fibroblasts used for experiments calcium-channel blockers. In order to clarify this cross- proliferated in the logarithmic phase between the 5th and susceptibility, we have isolated gingival fibroblasts from the 8th passage. patients reactive to nifedipine (seven strains), nicardipine (two strains), and nisoldipine (one strain), and tested the Cell-proliferation assay effect of calcium-channel blockers (nifedipine, diltiazem, The cell-proliferation assay was carried out by a nicardipine, and verapamil) and phenytoin on cell previously reported method (17). All ten strains of proliferation rate, DNA synthesis, collagen synthesis, and fibroblasts were subjected to the experiment in the following the number and Kd value of epidermal growth factor manner: Fibroblasts (approximately 3•~104) in 500 ƒÊl of (EGF) receptors in those gingival fibroblasts. Dulbecco's modified Eagle media (DMEM) supplemented with 10% fetal calf serum (FCS) and antimicrobial agents Materials and Methods (streptomycin 100 ƒÊg/ml, penicillin G 100 U/ml, and Cells amphotericin B 0.2 ƒÊgml, DMEM-10) were allowed to Cultures of fibroblast-like cells were established from settle in a 24-well plate (Falcon tissue culture plate 3047) gingival specimens of seven, two, and one patients who for 24 h. Cells were washed and the medium was replaced develop gingival overgrowth as a result of nifedipine , with DMEM containing 1% FCS and the same anti- nicardipine, and nisoldipine medications , respectively microbial agents as above (DMEM-1) for 24 h. Cells were (Table 1). All the specimens were obtained during again washed, a fresh 500 ƒÊ1 of DMEM-1 poured and 20 gingivectomy, clearance of remaining teeth, or orthopedic ul of 25 ƒÊM calcium-channel blockers (nifedipine, surgery of the alveolar ridge from the patient who gave diltiazem, verapamil, nicardipine; Sigma Chemical Co ., informed consent, under a protocol approved by the St. Louis, MO, U.S.A.) and phenytoin (Sigma Chemical Committee on Studies involving Human Beings of Nihon Co., St. Louis, MO, U.S.A.) were added to make the final University School of Dentistry at Matsudo. Disappearance concentration of 1ƒÊM in 4 wells each and kept for 48 h . or decreased severity of the overgrowth after discontinuance Our previous dose-response experiment revealed that 1 ƒÊM of medication was regarded as indicating a responsive of nifedipine gave the best growth in fibroblasts from state. Isolation and culture of gingival fibroblasts were nifedipine-reactive patients (17). Cells were then washed performed by the methods described previously (17,18). again and treated in the same manner as above except that Homogeneity of fibroblasts was determined by flow DMEM-10 was used.Cells were harvested using 0 .25% cytometry (FACS Vantage, Nippon Becton Dickinson Co. trypsin and 0.02% EDTA in Dulbecco's phosphate buffered Table 1 Source of cell strains 263 saline (DPBS) and the population count was performed ml centrifuge tube and then 1.5 ml of 10% TCA containing using a Coulter Counter ZM (Coulter Electronics, Ltd., 0.4% tannic acid and 0.0576% L-proline was added. The Luton, England). The number of cells in the absence of tube was centrifuged at 11,000 •~g for 3 min and the drugs served as the control. precipitated residue was washed three times with 750 ƒÊl of the same 10% TCA as above. The precipitates were then DNA synthesis washed with 750ƒÊl of ethylether:ethanol (1:3, v/v) and the DNA synthesis was determined using the method residue was air dried and dissolved in 300ƒÊl of 0.3 M NaOH previously reported (17) with a slight modification. at 37°C for 30 min. After neutralization with 300 ƒÊl of 0.3 Specifically, fibroblasts (approximately 6•~103 cells) in M HCl, the solution was divided into three portions (200 100 ƒÊl of DMEM-10 were allowed to settle in a 96-well ul each), the first portion being placed in a scintillation vial, the second portion digested by collagenase, and the third plate (Falcon tissue culture plate 3072) for 24 h. The adherent cells were washed and the medium was replaced portion acting as control for the second portion. Collagenase with DMEM-1 and then 4 ƒÊl of 25 ƒÊM calcium-channel digestion was performed by addition of 50 ƒÊlitube (400 blockers and phenytoin were added in the same manner U/tube) of collagenase (type VII, Sigma Chemical Co., St. as above except in 6 wells each and kept for 48 h. The Louis, MO, U.S.A.) solution dissolved in 1.4 M HEPES medium was changed again with DMEM-1 and calcium- buffer (pH 7.4) for 2 h at 37°C. The second and third channel blockers and phenytoin were added. 3H-thymidine portions were then treated with 650 ƒÊl of 10% TCA, centrifuged at 11,000 •~g for 3 min, and the supernatant (22.2 kBq, Amersham Life Science, Tokyo, Japan) was added to each well 24 h after the last medium change and was placed in the scintillation vial. All three fractions then incorporated into the cells for 24 h. During this pulse- were measured by liquid scintillation counting using label period, cells were in the S-phase stage and Ultima Gold (Packard Japan, Tokyo, Japan) as a scintillator. The radioactivities of the ones with non-drug treatment proliferation was very low because the medium (DMEM- 1) contained only 1% of FCS. After the treatment using served as the control. 0.25% trypsin and 0.02% EDTA in DPBS, cells were harvested with a cell collector (Micromate 196, Packard EGF receptor assay Japan, Tokyo, Japan), and the resulting radioactivities on EGF receptor assay was performed by the method the disc were measured using a direct beta counter (Matrix reported by King and Cuatrecasas (19) and Modeer et al. 96, Packard Japan, Tokyo, Japan). Radioactivity of the cell (20,21) with a slight modification. Specifically, fibroblasts in the absence of drug served as a control. (approximately 3•~104 cells) in 1.5 ml of DMEM-10 were allowed to settle in a 6-well plate (Falcon tissue culture Collagen synthesis plate 3046) for 24 h. The adherent cells were washed and Collagen synthesis was determined by the method the medium was replaced with DMEM-1 and then 60 ƒÊl described previously (17) with a slight modification. of 25ƒÊM calcium-channel blockers and phenytoin were Specifically, fibroblasts (approximately 3•~104 cells) in added in the same manner as above in 3 wells each and 500 ƒÊl of DMEM-10 were allowed to settle in a 24-well kept for 24 h.