Publication Date

2013

Document Type

Thesis

Committee Members

Steven Berberich (Advisor), Heather Hostetler (Committee Member), Michael Leffak (Committee Member)

Degree Name

Master of Science (MS)

Abstract

YPEL3 is a growth inhibitory gene that was established by our laboratory to trigger senescence in both primary and tumor cells. We recently reported YPEL3 to be directly repressed by estrogen in estrogen receptor-a positive (ER-a +ve) breast cancer cell lines. Here, we set out to determine whether the repression of YPEL3 involves histone deacetylation and, if so, elucidate the molecular mechanism of this activity. MCF-7, T-47D, and ZR-75.1 (all ER-a +ve) breast cancer cell lines were treated with varying doses of the global histone deacetylase inhibitors (HDACIs), trichostatin A (TSA) or suberoyl anilide hydroxamic acid (SAHA), for 24, 48 and 72 hours. Total RNA was isolated and the YPEL3 gene expression was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. Senescence was assayed using the senescence associated β-galactosidase (SA-β-gal) assay. RT-PCR analysis demonstrated an increase in YPEL3 expression in MCF-7 and T-47D cells treated with TSA or SAHA when compared to vehicle alone. In MCF-7 cells, this treatment correlated with a marked increase in cells staining positive for senescence for the 48- and 72- hour time points. The observed senescence was specific to YPEL3 as the treatment with HDACIs failed to induce comparable senescence in MCF-7 cells knocked-down for YPEL3. In this project, we establish and report one mechanism through which global HDACIs produce their effect on YPEL3, which is the down-regulation of ER-a mRNA and/or protein levels. RT-PCR analysis showed that of the two HDACIs, only SAHA reduces ER-a mRNA levels. In contrast, western blot experiments demonstrated a significant reduction of the ER-a protein levels in response to both TSA and SAHA following 48-hour treatment. This is consistent with our previous report of the inhibitory effect of estrogen, mediated by ER-a, on YPEL3 expression and associated senescence. Further investigation of a potential repressor complex recruited by ER-a to the estrogen response elements adjacent to YPEL3 promoter would provide answers as to whether HDACs directly participate in YPEL3 repression. The identification of epigenetic mechanisms involved in YPEL3 inactivation in ER-a positive breast cancer cell lines is critical for establishing treatment regimens that specifically target YPEL3 to induce senescence and limit tumor growth and spreading.

Page Count

101

Department or Program

Department of Biochemistry and Molecular Biology

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