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Our laboratory has developed and utilized an extensive human mammary epithelial cell (HMEC) culture system to study the proliferation barriers that prevent unlimited replication of cells derived from normal tissues. By examining one cell type, we have avoided potentially confounding variables due to species and cell type diversity. Our data support a model whereby HMEC encounter two mechanistically distinct proliferation barriers. The first barrier, stasis, is a consequence of accumulated stresses, and is mediated by the retinoblastoma (RB) pathway. Stasis is largely or fully telomere length independent. The onset of stasis in HMEC is correlated with increased levels of the CKI p16INK4a. In different culture conditions, HMEC can proliferate for 10-60 population doublings prior to elevation of p16 and proliferative arrest. In HMEC, stasis can be overcome by alterations in pathways governing RB (e.g., loss of p16 expression), and does not require loss of p53. A second, extremely stringent barrier, is imposed by critically shortened telomeres producing telomere dysfunction. Where wild-type p53 is present, this barrier has been termed agonescence, and produces a mostly viable growth arrest. If the second barrier is approached with non-functional p53, then crisis, rather than agonescence, occurs. Low levels of hTERT expression and telomerase activity can be detected in some pre-stasis HMEC, but not in post-stasis HMEC that encountered stasis and then overcame it, associated with silencing of p16. Our preliminary data suggest that HMEC that have not encountered stasis, or have bypassed stasis due to early inactivation of p16 by p16-siRNA, are more readily immortalized by agents that can elevate hTERT expression (e.g., c-myc) than HMEC that have encountered stasis. We hypothesize that RB-mediated stasis involves changes in chromatin conformation that may render the hTERT gene less accessible to transcriptional activation. We speculate that telomere-length independent stasis, and associated changes in chromatin, may be seen in other cell types arrested at what has been generically called senescence. However, cells may vary in perception and responses to specific stresses, with some cell types using p53-dependent p21, and/or p16 to prevent RB inactivation and to enforce stasis. We suggest that the use of molecularly defined nomenclature for the senescence barriers encountered by cultured human cells may facilitate understanding underlying mechanisms that are involved in carcinogenesis and tumor suppression.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]