Cooperative changes resulting in de-repression of telomerase and immortalization of human mammary epithelial cells. Free

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Unlimited proliferative potential (immortality) may be required to accumulate the multiple rare errors necessary for malignancy and metastasis. In most human somatic cells, telomerase activity is low or undetectable and telomere dysfunction provides a stringent limit to the number of replications a single lineage can undergo, thus limiting the opportunity for deleterious mutations to accumulate. Human tumor cells may be derived from cells in which telomerase has been de-repressed through genetic and/or epigenetic events. Although a number of telomerase regulators have been proposed, the regulators whose alterations during carcinogenesis play causative roles in telomerase de-repression are still unknown. To define a pathologically relevant model of human mammary epithelial cell (HMEC) immortalization, we have exposed telomerase(-) cells cultured from normal tissue to the potential immortalizing oncogenes ZNF217 and c-MYC. We assessed the following criteria at different passages following retroviral infection: a) transgene expression, b) hTERT mRNA expression, c) telomerase activity, d) mean TRF length, e) colony forming efficiency and labeling index ± TGFbeta, f) p57Kip2 expression, g) genome copy number changes (by CGH), and h) cell morphology. Although transduction of additional copies of the ZNF217 gene alone often resulted in immortal cells, telomerase de-repression occurred gradually and additional genomic changes were detected in the immortal cells. Transduction of the c-MYC gene alone rarely resulted in immortal cells. To determine whether the ZNF217 and c-MYC genes can act cooperatively to yield more efficient immortalization of HMEC, the genes were transduced together into growing HMEC, and independently infected populations were monitored. In some populations, the combination yielded immortal lines without a significant slowdown in growth. Three such lines also did not show large genome copy # changes by array CGH. However, only some populations yielded this result, and those that did showed limited numbers of retroviral insertion sites, indicating that additional genetic/epigenetic events may be necessary for immortalization. These studies have led us to propose a model in which at least two alterations are required for de-repression of endogenous telomerase activity. If one predisposing alteration is already present, e.g., deregulated ZNF217 or c-MYC, the probability greatly increases that the required additional error(s) may be generated by the genomic instability produced by telomere dysfunction. Augmentation of the processes that monitor and prevent growth of cells with dysfunctional telomeres may be useful for prevention of acquisition of telomerase activity and further malignant changes.