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

Abstract

The in vitro 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 beta-galactosidase (SA-beta-gal). Subsequently, with the increase in cell passages, the copy number of the exogenously transfected hTERT gene and the percentage of SA-beta-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 p21(WAF) 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. p16(INK4a) 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 p16(INK4a) 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 in vitro immortalization in HME cells may require the activation of the function of telomerase and other genetic alterations such as the spontaneous loss of p16(INK4a) expression.

Alterations in the p16INK4a and p53 tumor suppressor genes of hTERT-immortalized human fibroblasts

“…INK4a pathway is inactivated (Kiyono et al, 1998;Farwell et al, 2000;Kim et al, 2002;Toouli et al, 2002). Likewise, for some strains of lung (MacKenzie et al, 2000;Franco et al, 2001), mammary (O'Hare et al, 2001) and periodontal ligament (Tsutsui et al, 2002) fibroblasts, hTERT-transduced cells required additional mutational events including the loss of p16…”

Cellular characteristics of primary and immortal canine embryonic fibroblast cells

“…Transcriptional silencing of p16 INK4a in many immortal cells is due to the hypermethylation of its promoter (Merlo et al, 1995;Serrano, 1997). As shown in Figure 4A, in the CGFR-Ca-1 and -2 cell lines, expression of canine p16 INK4a mRNA was shown to be evidently detectable and was not even altered by treatment with DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC) (Kim et al, 2002b). However, p16 INK4a mRNA was found to be dramatically decreased, while restored in immortal CGFR-Ca-3 Relative cell growth (%)…”

“…Common genetic alterations frequently observed in the immortalized cells are either associated with the loss of p53 and Rb regulatory functions that are linked with cell-cycle arrest and cell death or the activation of telomerase that maintains telomere integrity or both (Bodnar et al, 1998;Sherr and DePinho, 2000;Kim et al, 2002b).…”

Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells

“…Several studies have demonstrated the possibility of establishing immortalized human cell lines in culture. Expression of the catalytic subunit of telomerase (hTERT) could prevent growth arrest and immortalize human fibroblasts and epithelial cells (Morales et al, 1999;Kim et al, 2002b). While these immortalized cells actively proliferated in culture, their growth requirements, cellcycle checkpoints and karyotypic stability were similar to the normal parental cells.…”