Publication Date


Document Type


Committee Members

Gerald Alter (Committee Member), Steven Berberich (Advisor), Michael Markey (Committee Member)

Degree Name

Master of Science (MS)


p53 activation through different cellular senescence pathways can trigger cell cycle arrest via regulation of p53 target genes. One such target gene is YPEL3 which is expressed upon binding of tumor suppressor protein p53 at its p53 binding sites (Kelley, 2010). The ability of p53 to induce YPEL3 gene expression led to the discovery that YPEL3 is one of several p53 target genes which induce cellular senescence (Kelley, 2010). Additionally YPEL3 can be regulated independently of p53 by estrogen signaling through estrogen receptor α (Tuttle, 2011). The loss of estrogen receptor α or removal of estrogen induces YPEL3 gene expression and leads to cellular senescence, indicating that estrogen bound to estrogen receptor α represses YPEL3 gene expression (Tuttle, 2011). Although YPEL3 induction results in cellular senescence the mechanism by which YPEL3 elicits cellular senescence is not well understood. It is also unknown if other steroid hormones, such as testosterone play a role in regulating YPEL3 gene expression To further understand hormone regulation of YPEL3 the first part of this thesis tested if testosterone regulates YPEL3 gene expression in MCF7 breast cancer cells and LnCAP prostate cancer cells. Like MCF7 breast cancer cells, LnCAPs cultured in the absence of steroid hormones induced YPEL3 expression indicating that YPEL3 gene expression in LnCAPs is repressed by steroid hormones. This induction of YPEL3 expression was blocked by the addition of testosterone to LnCAP cells. In contrast the addition of testosterone to steroid deprived MCF7 cells resulted in YPEL3 induction. Based on the results in LnCAP prostate cancer cells and MCF7 breast cancer cells it appears that testosterones effect on YPEL3 gene expression is tissue specific. In part two of this thesis MCF7 and IMR90 cells were employed to determine if over expression of YPEL3 leads to increased reactive oxygen species (ROS) levels. First an optimized method for detecting reactive oxygen species levels in breast cancer cells using DCFDA was developed. Utilizing this method, MCF7 human breast cancer cells harboring a Tet-On system expressing YPEL3 induced with tetracycline did not show increased levels of reactive oxygen species over LacZ expressing MCF7 cells. Additionally Infecting MCF7 cells with lentivirus expressing YPEL3 and probing with DCFDA showed no increase of ROS levels. Alternatively IMR90 primary diploid human fibroblasts containing a normal repertoire of genes and fully functional pathways were infected with lentivirus expressing YPEL3 and also did not show an increase in ROS levels. These results suggest that YPEL3 activates senescence in a ROS independent manner. The third part of this thesis was to identify YPEL3 interacting proteins. Epitope tagged YPEL3 proteins obtained from MCF7 tetracycline responsive cells expressing YPEL3 were captured from cell extracts by co-immunoprecipitation, followed by elution and denaturing. Denatured proteins were separated by SDS-Page gel electrophoresis and potential protein bands excised for composition analysis by LC/MS/MS. LC/MS/MS analysis identified potential proteins that interact with YPEL3. The cumulative findings of this thesis were designed to aid in the understanding of YPEL3 regulation by testosterone and to assist in locating potential downstream targets of YPEL3 that may lead to senescence.

Page Count


Department or Program

Department of Biochemistry and Molecular Biology

Year Degree Awarded