P16/p53 expression and telomerase activity in immortalized human dental pulp cells

Introduction: Residing within human dental pulp are cells of an ectomesenchymal origin that have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence. This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells (nDPSCs). Results: With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP, COL-1, DMP-1, DSPP, OCN and OPN), as did nDPSCs. Oncoprotein expression was seen in both groups except for a noted absence of p16 in the tDPSCs. nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed as well as low telomerase activity readings. Methods: Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured and their telomerase activities were determined using a telomerase quantification assay. Also examined, were the expression of genes involved in proliferation and mineralization such as human alkaline phosphatase (ALP), beta-actin, collagen I (col-1), core binding factor (cbfa)-1, dentin matrix protein (DMP-1), dentin sialophos-phoprotein (DSPP), GAPDH, hTERT, osteocalcin (OCN), osteopontin (OCN), as well as oncoproteins involved in senescence (p16, p21 and p53) using RT-PCR. DNA and alkaline phosphate activity was also assayed in both cell groups. Conclusion: These results indicate maintenance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression.