Induction of Apoptosis in Senescent Gingival Fibroblasts

Objective: The effect of apoptosis-inducing signals on human gingival fibroblasts has not been studied adequately. The purpose of this study was to examine the effects of apoptosis-inducing molecules on rapidly dividing and senescent gingival fibroblasts.

Methods: Human Gingival Fibroblasts from a primary cell line (from tissue without periodontal disease) underwent a series of passages until they reached replicative senescence. Cells from passage 16 were considered as rapidly-diving cells whereas cells from passage 33 were considered to be senescent. Both senescent and rapidly dividing cells were treated with increasing concentrations of either Tumor necrosis factor-α (TNF-α), Ceramide, Staurosporine and Hydrogen Peroxide (H2O2). Hoechst 33342 and propidium iodide were used to stain the cells and reveal apoptotic and necrotic cells. Counts of alive, apoptotic and necrotic cells were taken at 2, 4 and 24h.

Results: Ceramide and staurosporine induced apoptosis when added on both rapidly-diving and senescent HGFs. In both cases, senescent cells were induced to become apoptotic by lower concentrations of these drugs than did their rapidly dividing counterparts. In addition, H2O2 induced apoptosis in senescent cells, but not rapidly dividing cells. However, the transition from apoptosis to necrosis seems to be delayed in the GFp33 (50% necrosis after 24 hours, vs. 100% for GFp16). TNFα had no effect on HGFs. Thus, senescent cells were more sensitive to Ceramide, staurosporine and H2O2 than rapidly-diving cells.

Conclusion: Senescent gingival fibroblasts seem more sensitive to apoptosis, but the transition from apoptosis to necrosis may be delayed in these cells. These changes may imply a role of cellular senescence in determining increased susceptibility to apoptosis in gingival tissues with aging.

Supported by NIH P20RR20160.