Immortal potential is thought to be crucial for malignant progression during human breast carcinogenesis. In our laboratory, cultured human mammary epithelial cells (HMEC) are used to study changes accompanying and contributing to immortalization. HMEC immortalized by a variety of agents (the chemical carcinogen benzo[a]pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up-regulation (11-24-fold) of the telomere-binding protein, TRF2. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We also found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite lifespan and immortal HMEC. However, blockage of translation with cycloheximide did not reveal differences in protein stability. Immunofluorescence studies of TRF2 abundance indicated considerable heterogeneity in TRF2 expression within a single clonally derived immortal HMEC line. This heterogeneity does not appear to be due to cell cycle status. We are currently investigating a) whether nuclear localization and levels of expression of other telomeric proteins are affected by the altered TRF2 levels; b) potential causes of increased accumulation of TRF2 in the nuclei of fully immortal HMEC, and c) the consequences of TRF2 over-expression. We speculate that the processes responsible for increased TRF2 expression in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells’ ability to maintain an indefinite lifespan. Identifying the causes and consequences of TRF2 over-expression may clarify the changes occurring during early breast carcinogenesis.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]