Microarray analysis of in vitro senescence of human gingival fibroblasts

Objectives: Cellular or replicative senescence is the phenomenon where normal diploid differentiated cells lose the ability to divide. Some evidence exists to suggest a relationship between cellular senescence and aging. As gingival fibroblasts reach senescence, they incur hundreds of biological changes that affect virtually all of their activities. DNA microarrays allow for the simultaneous analysis of gene expression patterns of whole genomes. The purpose of this study was to examine the senescence of gingival fibroblasts using microarray technology.

Methods: Gingival fibroblast cultures were established from explants of healthy gingiva obtained from patients who underwent periodontal surgery. Gingival fibroblasts grown for 16 passages (16P) were compared to senescent cells that underwent 33 passages (33P). Transcript expression in gingival samples from these two groups was compared using microarray analysis (U133 Plus 2.0, Affymetrix). Differential expression of transcripts was confirmed using RT-PCR.

Results: Cell division rates remained nearly constant through passage 30, at which time cell division rapidly slowed. Of the 54,000 probe sets examined, 613 genes displayed at least 2-fold difference at level of significance 0.05 (P16 cells expressed 203 genes at higher levels, while P33 cells expressed 410 genes at higher levels). Among these differentially expressed genes, several classes of molecules were identified, including; attachment and extracellular matrix proteins, cytokines and growth factors, cyclins, histones and genes involved in apoptosis.

Conclusions: Cellular senescence of gingival fibroblasts results in changes in the expression of numerous genes compared to rapidly dividing gingival fibroblasts. These changes may imply a role of cellular senescence in determining increased susceptibility to periodontal disease with aging.