- Paper Report
- Open Access
An in vitrobreast cell immortalisation assay
- Jenny Gomm1
© Biomed Central Ltd 2001
- Received: 1 May 2001
- Accepted: 20 August 2001
- Published: 1 December 2001
- Chemopreventive agent, immortalization, Li-Fraumeni syndrome, telomerase
In most normal human cells there is a progressive shortening of chromosomal telomeres with successive cell divisions, leading ultimately to cell senescence. Cellular immortalisation is thought to occur by the reactivation of a telomerase which prevents telomere shortening and consequent growth arrest. As telomerase activity has been detected in 90% of cancers tested, it may be a good target in cancer treatment. Early-onset breast cancer is prevalent amongst patients with Li-Fraumeni syndrome (LFS). In previous studies, the authors demonstrated that normal breast epithelial cells from women with LFS immortalise spontaneously in culture. Normal breast epithelial cells were used in an in vitro assay to investigate the effects of chemopreventive and antitelomerase agents on telomerase activity and the immortalisation process.
Treatment of breast epithelial cells with established chemopreventive agents, such as oltipraz, tamoxifen, 9-cis-retinoic acid, 13-cis-retinoic acid and difluoromethylornithine, resulted in fewer spontaneous immortalisation events than those compared with solvent-treated controls. This treatment was only significant, however, for treatment with tamoxifen, 9-cis-retinoic acid and 13-cis-retinoic acid. Transfection with antisense or dominant-negative telomerase RNA had a similar inhibitory effect on the incidence of spontaneous immortalisation. Telomerase activity was absent in all preimmortal cells and present in all clones of spontaneously immortalised cells.
Cell culture, retroviral infection, transfection, PCR, fluctuation analysis
Cells from LFS patients may be difficult to come by and human mammary epithelial cells, which are commercially available, may provide a useful alternative. These have spontaneously immortalised in culture but whether they exhibit a sufficient frequency of events would need to be studied.
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