Disruption of the retinoblastoma pathway by small interfering RNA and ectopic expression of the catalytic subunit of telomerase lead to immortalization of human ovarian surface epithelial cells

Abstract

The risk of developing ovarian cancer is about 1% over a lifetime, but it is the most deadly gynecologic cancer, in part due to lack of diagnostic markers for early-stage disease and cell model system for studying early neoplastic changes. Most existing immortal human ovarian surface epithelial cells were achieved by using viral protein such as SV40 T/t antigen or E6/E7, which inactivate multiple cellular pathways. In the current study, we used a small interfering RNA (siRNA) against the retinoblastoma gene (pRb) and ectopic expression of human telomerase reverse transcriptase (hTERT) to immortalize the primary ovarian epithelial cell line OSE137 and two additional human ovarian surface epithelial cells. The immortalized OSE137 showed increased telomerase activity, lengthened telomeres, increased G 2 /M phase, altered cell-cycle regulatory proteins but nontumorigenic. As both Rb and hTERT pathways are commonly altered in human ovarian cancer and these genetic changes are faithfully modeled in these cells without using viral protein, these immortal cells represent an authentic in vitro model system with which to study the initiation and progression of human ovarian cancer.

A novel function of colony-stimulating factor 1 receptor in hTERT immortalization of human epithelial cells

“…Earlier studies have shown immortalization of human epithelial cells through disruption of pRb and/or p53 function in combination with overexpression of hTERT (Whitaker et al, 1995;Hahn, 2002;Yang et al, 2006). However, we and others have described earlier that the only step required for the immortalization of somatic cells is activation of telomerase (Jiang et al, 1999;Morales et al, 1999;Li et al, 2007).…”

Telomerase in the ovary

“…To establish whether a single disruption of either the p53 or RB1 pathway would promote telomerase-induced human OSE cell immortalization, Liu et al used RNAi technology to knock down either RB1 or p53 in combination with ectopic expression of the TERT in human OSE cells. Significantly, knockdown of either RB1 (Yang et al 2007a) or p53 (Yang et al 2007b) in the presence of TERT is sufficient to immortalize human OES cells. It was later shown that human OSE cells could be immortalized by TERT expression without functional disruption of RB1 and p53 (Li et al 2007).…”

“…Thus, while telomerase activity is indispensable in the immortalization step of human OSE, p53 and RB1 are not, at least under certain conditions. Furthermore, TERT-immortalized human OSE cells have lengthened telomeres, are diploid without chromosomal instability, and maintain epithelial characters without tumorigenicity (Kusakari et al 2003, Yang et al 2007a, 2007b, Sasaki et al 2009). …”

“…Work from several independent laboratories has shown that forced expression of the telomerase reverse transcriptase (TERT) gene to maintain telomeres in cultured OSE cells results in increased proliferative potential of the cells, causing them to resemble stem cells (Davies et al 2003, Kusakari et al 2003, Alvero et al 2004, Maeda et al 2005, Li et al 2007, Yang et al 2007a, 2007b. This fundamental effect of telomere remodeling on OSE cells demonstrates proof of principle that the regulation of telomere remodeling is closely linked to OSE cell proliferative capacity.…”

Ovarian cancer: pathology, biology, and disease models

“…To overcome this limitation, we generated a second-generation model of immortalized, nontumorigenic cells by using retrovirus-mediated small-interfering RNA against p53 or Rb, in combination with the ectopic hTERT expression (124–125). This new model offers new opportunities to study the mechanisms underlying development of different types and grades of ovarian cancer and to define the number and combinations of genetic changes required for ovarian carcinogenesis.…”