Abstract 1230: Detection of increased O-GlcNAcylated p53 levels in MCF-7 cells after O-GlcNAcase inhibitor treatment using antibodies specific for Ser-149-O-GlcNAcylated p53 proteins
Authors: Kim Hyesook , KIm So Hee , Joiakim Aby , Kaplan David , Bae Sung Hun , Putt David 
Published: August 2015

Abstract
Protein O-GlcNAcylation (N-acetylglucosamine modification) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. Most serine (Ser) and threonine (Thr) sites for O-GlcNAcylation are on or near to phosphorylation sites, which creates a system of mutual elimination. O-GlcNAcylation of p53, a tumor suppressor protein, at Ser-149 prevents p53 degradation and stabilizes p53 proteins expressed in MCF-7 human breast cancer cells whereas phosphorylation of the p53 at Thr-155 induces ubiquitin-dependent degradation, which decreases p53 levels. Accumulation of the p53 protein in cancer cells induces arrest of cell growth and apoptosis in response to DNA damage. Ser-149 site-specific anti-peptide antibodies have been produced using KLH conjugated with p53 peptides with and without O-GlcNAcylation at Ser-149 by immunization of rabbits for polyclonal and mice for monoclonal antibodies. Whereas polyclonal antibodies and hybridoma media for O-GlcNAcylated p53 cross-reacted with BSA conjugated with Ser-149-O-GlcNAcylated p53 peptides in ELISA or Western blot analysis, the antibodies did not cross-react with BSA conjugated with p53 peptides without O-GlcNAcylation. Anti-peptide polyclonal antibodies produced for the unmodified p53 peptides did not cross-react with BSA conjugated with Ser-149 O-GlcNAcylated p53 peptides. Using the hybridoma media for Ser-149-O-GlcNAcylated p53, Western blot analysis was carried out with nuclear lysates and cytosol-containing supernatant obtained from MCF10A (normal breast cancer cell line) and MCF-7 cells. It was found that, in MCF-10A cells, Ser-149-O-GlcNAcylated p53 proteins (∼55 kDa) were minimally expressed in the nuclear fraction but highly expressed in the cytosolic fraction whereas, in MCF-7 cells, the level of cytosolic Ser-149-O-GlcNAcylated p53 protein was low compared with the level in MCF-10A cells. MCF-7 cells were treated for 24 hr with 1% DMSO or 2 mM β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, acetazolamide, dissolved in DMSO and levels of Ser-149-O-GlcNAcylated p53 were determined by Western blot analysis using antibodies for the Ser-149-O-GlcNAcylated p53. Whereas the level of O-GlcNAcylated p53 did not increase after 1% DMSO treatment, the level dramatically increased after treatment of acetazolamide in 1% DMSO and induced cell death. Our results showed that acetazolamide treatment increased Ser-149-O-GlcNAcylated p53 level and induced cell death in MCF-7 cells, which may decrease invasiveness of the breast cancer cells. Supported by NCI SBIR Phases I and II Contracts No. N261201100073C and N261201300058C.
Citation Format: Hyesook Kim, So Hee KIm, Aby Joiakim, David Kaplan, Sung Hun Bae, David Putt. Detection of increased O-GlcNAcylated p53 levels in MCF-7 cells after O-GlcNAcase inhibitor treatment using antibodies specific for Ser-149-O-GlcNAcylated p53 proteins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1230. doi:10.1158/1538-7445.AM2015-1230