Relationship between Gingival Overgrowth and Lipopolysaccharide Induced Apoptosis

Objectives: It has previously been demonstrated that gingival fibroblasts derived from nifedipine-reactive patients (NIFrs) showed a greater cell proliferation rate than those from nifedipine non-reactive patients (NIFns) in the presence of 1μM nifedipine. It has also been shown that gingival overgrowth was caused through inhibition of apoptosis by nifedipine, and apoptosis of fibroblast was induced by lipopolysaccharide (LPS). The tight regulation of apoptosis is modulated by various factors such as death gene p53 and survival gene bcl-2. Thus, we focused the apoptotic effect of LPS on NIFn and NIFr cells. Methods: Cells were cultured until semi-confluent in DMEM-10, arrested in DMEM-0.5 for 24 hrs, and then stimulated by various concentration of LPS (1, 3, 6, 10, and 30 μg/ml) for 24 hrs. After stimulation by LPS, number of apoptotic cells in NIFn and NIFr cells was measured using APOPercentage^TM Apoptosis Assay Kit and CycleTEST^TM PLUS DNA Reagent Kit. For analysis of apoptosis regulating genes expressions in NIFn and NIFr cells, cells were stimulated by 10 μg/ml of LPS for 6, 12, and 24 hrs, and then total RNA was isolated and gene expressions were analyzed using RNeasy Mini Kit and OneStep RT-PCR Kit, respectively. Results: Apoptotic cells in NIFn were significantly increased by 10 and 30 μg/ml of LPS, but those in NIFr were seldom. The p53 and bcl-2 mRNA expressions were found in NIFn cells at 6, 12, 24 hrs after stimulation by 10 μg/ml LPS, but not in NIFr cells. Conclusion: Since apoptosis plays a key role in cellular response to DNA damage, decrement of apoptotic cells in NIFr cells may relate to the cause of gingival overgrowth. This investigation was supported in part by MEXT, Academic Frontier Project (2003-2007; AF, HM, YA) in Japan.