Access Type

Open Access Dissertation

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Michael A. Tainsky

Abstract

Bypassing cellular senescence, an irreversible growth arrest of cells that is activated in normal cells to become immortal is one of the prerequisites for carcinogenesis. Cellular senescence can be triggered by shortening of telomeres and certain cellular stresses. Using spontaneously immortalized Li-Fraumeni Syndrome (LFS) fibroblasts, we found that CREG1 (Cellular Repressor of E1A-stimulated Genes1) is one of genes whose expression fit the criteria of senescence-associated genes, decreased expression during immortalization and increased in senescence. Moreover, we found that epigenetic mechanisms regulate CREG1 expression in LFS fibroblasts. CREG1 is a secreted glycoprotein that was shown to bind Rb-family pocket proteins and inhibit E2F transactivation activity. Therefore, we hypothesize that CREG1 plays a role in cellular senescence involving the p16INK4a-Rb pathway. Ectopic expression of CREG1 in the immortal LFS cell lines decreases cell proliferation but does not directly induce senescence. We confirmed this in osteosarcoma and fibrosarcoma cancer cell lines, similar to those seen in patients with Li-Fraumeni Syndrome. Because CREG1 was shown to interact with Rb pocket proteins in vitro, we tested whether growth suppression by CREG1 may depend on the phosphorylation status of Rb. We found that p16INK4a is also downregulated in immortal cells and that coexpression of CREG1 and p16INK4a, an inhibitor of CDK and Rb phosphorylation, has a greater effect than CREG1 or p16INK4a alone to reduce cell growth, to induce cell cycle arrest and cellular senescence in immortal LFS, osteosarcoma and fibrosarcoma cell lines. Moreover, cooperation of CREG1 and p16INK4a inhibits the expression of cyclin A and cyclin B by inhibiting promoter activity thereby decreasing mRNA and protein levels; these proteins are required for S-phase entry and G2/M transition. In conclusion, we are the first to find that CREG1 enhances p16INK4a-induced senescence by transcriptional repression of cell cycle mediated genes.

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