Abstract LB-58: Production of Thr-58-O-GlcNAcylated c-myc antibodies and detection of increased O-GlcNAcylated c-myc levels in MCF-7 cells after ketoconazole treatment.

Abstract

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. Thr-58 of c-myc, a mutational hot spot in lymphomas, is a site for both phosphorylation (primed by Ser-62 phosphorylation) and O-GlcNAcylation. Whereas Thr-58 O-GlcNAcylation induces ubiquitin-dependent c-myc degradation, Thr-58/Ser-62 phosphorylation increases c-myc stability and invasiveness and tumorigenesis of MCF-7 human breast cancer cells. Thr-58 site-specific anti-peptide antibodies have been produced by immunization of rabbits with KLH conjugated with c-myc peptides with and without Thr-58 O-GlcNAcylation. Whereas the antibodies for O-GlcNAcylated c-myc cross-reacted with BSA conjugated with Thr-58-O-GlcNAcylated c-myc peptides in ELISA and Western blot analysis, the antibodies did not cross-react with BSA conjugated with c-myc peptides without O-GlcNAcylation. Anti-peptide antibodies produced for the unmodified c-myc peptides did not cross-react with BSA conjugated with Thr-58-O-GlcNAcylated c-myc peptides. Using the antibodies for Thr-58-O-GlcNAcylated c-myc, Western blot analysis was carried out with nuclear lysates and cytosol-containing supernatant obtained from MCF-7 cells and it was found that Thr-58-O-GlcNAcylated c-myc proteins were minimally expressed in the cytosolic fraction but highly expressed in the nuclear fraction. Whereas c-myc proteins without Thr-58-O-GlcNAcylation in the nuclear fraction were detected primarily as 65 kDa proteins in Western blot analyses, Thr-58-O-GlcNAcylated c-myc proteins in the nuclear fraction were detected primarily as 68 kDa proteins. MCF-7 cells were treated for 4 hr with 1% DMSO and 2 mM β-N-acetylglucosaminidase (O-GlcNAcase) inhibitors (buspirone or ketoconazole) dissolved in DMSO and levels of Thr-58-O-GlcNAcylated and unmodified c-myc were determined by Western blot analysis using antibodies for the Thr-58-O-GlcNAcylated or unmodified c-myc. Whereas the O-GlcNAcylated c-myc level did not increase after buspirone treatment, the level dramatically increased after ketoconazole treatment. The Thr-58-unmodified c-myc protein level failed to increase after 2 mM ketoconazole treatment. The ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells as evidenced by lactate dehydrogenase (LDH) cytotoxicity assays. Our results showed that ketoconazole treatment increased Thr-58-O-GlcNAcylated c-myc level and induced cell death in MCF-7 cells, which may decrease invasiveness of the breast cancer cells. Supported by NCI SBIR Phase I Contract No. HHSN261201100073C. Citation Format: Hyesook Kim, Sohee Kim, Aby Joiakim, David Kaplan, David Putt. Production of Thr-58-O-GlcNAcylated c-myc antibodies and detection of increased O-GlcNAcylated c-myc levels in MCF-7 cells after ketoconazole treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-58. doi:10.1158/1538-7445.AM2013-LB-58 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.