Gene expression pathways altered in cellular immortalization

Cellular immortalization is a necessary step in the evolution of a cell as it progresses to become a tumor. Cellular senescence is a property of normal cells that must be abrogated for immortalization to occur. Four independent spontaneously immortalized Li-Fraumeni syndrome (LFS) cells lines were used to analyze the nature of the genetic changes that may have given these cell lines the growth advantage required to become immortalized. A cellular senescence-like phenotype can be induced in immortalized LFS cells by treatment with the DNA methyl-transferase inhibitor 5-aza-deoxycytidine, suggesting that the immortal phenotype of these cells is reversible. We hypothesized, that genes epigenetically silenced by promoter methylation could be key regulators of senescence. We used microarrays to evaluate epigenetic control of gene expression in immortal LFS cells. Fourteen genes that were epigenetically repressed during immortalization in all four immortal LFS cells lines were identified. Additionally gene ontology analysis of the expression data revealed a statistically significant contribution of interferon pathway, 324 cell cycle and cytoskeletal genes in the process of immortalization. The role of individual genes including IGFBPrP1, one of the 14 epigenetically regulated, 2 related genes, IGFBP3 and IGFPB4, and a cytoskeletal gene, testin (TES), were further assessed. The expression of these genes increased in senescent MDAH041 cells. IGFBP family genes are capable of inhibiting cell proliferation and can induce apoptosis in cancer cells. We found overexpression of IGFBP3 or IGFBPrP1 in immortal LFS cell lines inhibited cell growth. The cytoskeletal gene TES was analyzed because it fits the profile of a senescence-regulating gene: silenced during immortalization and reactivated by DNA demethylation. Bisulfite sequencing confirmed that silencing of TES in the LFS cells is due promoter CpG island methylation. Overexpression of TES resulted in a reduction in cell proliferation and cell senescence. Elucidating how a cell is able to bypass replicative senescence and become immortal has provided new insight into molecular mechanions that can be targeted so as to reverse carcinogenesis at an early stage.