Extracts of Actinobacillus actinomycetemcomitans induce apoptotic cell death in human osteoblastic MG63 cells

Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteobla stic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T 3-E1 cells in the presence of such extracts. The addition of the extracts f rom Actinobacillus actinomycetemcomitans induced cell death in MG63 cells i n a dose- and time-dependent fashion over the concentration range of 0.1 to 10 mu g/mL. By contrast, the extracts from Prevotella intermedin did not i nduce cell death in these cells, even in the presence of 10 mu g/mL protein . By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extract s from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder form ation was dose-dependent with a maximal effect at a concentration of 10 mu g/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Pre votella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3 T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Soni cated extracts of Actinobacillus actinomycetemcomitans that had been treate d with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cyclohexi mide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new ge ne transcription and protein synthesis are regulated for Actinobacillus act inomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate th at apoptosis in alveolar bone cells induced by Actinobacillus actinomycetem comitans plays an important role in periodontal diseases.