Effects of Chemopreventive and Antitelomerase Agents on the Spontaneous Immortalization of Breast Epithelial Cells

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Abstract
Background: Activation of telomerase is an early event in the development of breast and other cancers that may lead to cell immortalization, a critical and rate-limiting step in cancer progression. Breast epithelial cells from women with Li–Fraumeni syndrome (LFS) immortalize spontaneously and reproducibly in culture. We, therefore, tested whether immortalization of these cells could be prevented by treating them with chemopreventive agents and by inhibiting telomerase activity. Methods: Noncancerous, preimmortal breast epithelial cells derived from a patient with LFS were treated for 3 months with nontoxic concentrations of the chemopreventive agents oltipraz, difluoromethylornithine, tamoxifen, and retinoic acid or with two different telomerase inhibitors. The frequency of spontaneous immortalization of LFS-derived cells was estimated by an approach based on fluctuation analyses. Statistical analyses were two-sided. Results: The frequency of spontaneous immortalization events of LFS-derived breast epithelial cells was reduced by long-term treatment with retinoic acid (P<.001) or tamoxifen (P<.05) compared with solvent-treated cells. The frequency of immortalization was also reduced by treating LFS-derived cells with an antitelomerase antisense oligonucleotide (P<.001) or by inducing the cells to express a dominant negative mutant of telomerase (P<.025) compared with cells treated with a control oligonucleotide or with empty vector, respectively. Conclusions: Treatment of preimmortal LFS breast epithelial cells with chemopreventive and antitelomerase agents decreased the frequency of spontaneous immortalization in vitro. These studies validate the application of a new cell culture model system to screen the effects of novel chemopreventive agents by use of cell immortalization as an end point. The results also suggest that the telomerase ribonucleoprotein complex may be an important molecular target for breast cancer prevention.