Characterization of immortalized human gingival fibroblasts by hTERT

Objectives: To immortalize human gingival fibroblasts (HGFs) by hTERT retroviral infection and to characterize immortalized-HGFs (I-HGFs) as a model of role of HGFs in oral diseases.

Methods: I-HGFs were characterized by cell morphology, proliferation analysis, cell-cycle-analysis, colony-formation-assays, beta-galactosidase-staining, gene-expression analysis by Affymetrix-U133plus2-microarrays, and by Q-RT-PCR.

Results: HGFs immortalized by hTERT and selected with G418 did not change their morphology compared with normal-HGFs. Microarray analysis and Q-RT-PCR results revealed up-regulation of hTERT gene in I-HGFs. Cell proliferation studies exhibited similar patterns in normal and immortalized cells. Cell-cycle analysis indicated an increase in S and G2 phase in I-HGFs. Colony-formation-assays showed that I-HGF cells, like normal-HGFs, did not form colonies whereas positive control cells (tumorogenic mouse SSV NIH 3T3 cells), formed colonies indicating that hTERT-cells are not transformed. Gene-expression-analysis showed down-regulation in cyclin A2, B1, B2, CDCA3, CDCA8, CDKN3, FOXM1, UBE2C, FGF5, STK6, HAS2 and NEK2 genes in beta-gal positive senescent-cells compared with normal-HGFs, however these expression levels were not significantly different in immortalized cells. Cyclin D2 was up-regulated in senescent-cells but remained normal in immortalized-cells. Cyclin D1 was normal in senescence and down-regulated in immortalized-cells compared to normal-HGFs. PERP, GAS6, PRKCDBP, SOD2, AK3L1, TGFB2 genes were normal in senescence and down-regulated in the immortalized-cells showing that these genes might be the targets of hTERT regulation. I-HGF cells have been maintained for over 65 passages without observable changes in phenotype compared to normal cells which undergo senescence at passage ~10-15.

Conclusion: Immortalization of HGFs exhibited extended normal growth potential without transforming and exhibited a limited range of expression changes largely related to cyclin and cyclin-related-molecules. These results reveal that immortalization by hTERT is associated with changes in cell-cycle regulation. I-HGFs are likely a useful model of HGF properties in gingival inflammation. NIH/CA114810.