Events in the immortalizing process of primary human mammary epithelial cells by the catalytic subunit of human telomerase

摘要

pThe iin vitro/i immortalization of primary human mammary epithelial (HME) cells solely by the exogenous introduction of the catalytic subunit of human telomerase (hTERT) has been achieved. Early passage hTERT-transfected HME (T-HME) cells continuously decreased the length and density of telomeres even in the presence of telomerase activity, with a significant number of cells staining positive for senescence-associated β-galactosidase (SA-β-gal). Subsequently, with the increase in cell passages, the copy number of the exogenously transfected hTERT gene and the percentage of SA-β-gal positive cells were found to decrease. Eventually, a single copy of the exogenous hTERT gene was observed in the relatively later passage T-HME cells in which telomere length was elongated and stabilized without obvious activation of endogenous hTERT and c-Myc expression. In T-HME cells, the expression of two p53 regulated genes p21supWAF/sup and HDM2 increased (as in primary senescent HME cells), and was found to be further elevated as the function of p53 was activated by treatment with DNA-damaging agents. p16supINK4a/sup was shown to be significantly higher in the primary senescent HME and the early passage T-HME cells when compared with the primary presenescent HME cells, with a dramatic repression of p16supINK4a/sup observed in the later passage T-HME cells. In addition, the expression of E2F1 and its transcription factor activity were found to be significantly higher in the later passage T-HME cells when compared with the earlier passage T-HME cells. Together, our results indicate that iin vitro/i immortalization in HME cells may require the activation of the function of telomerase and other genetic alterations such as the spontaneous loss of p16supINK4a/sup expression./p