Abstract 1028: Regulation of glucocorticoid-evoked apoptosis in human leukemic CEM cells by the C. elegans ces2 ortholog E4BP4

Abstract

Abstract Identification of regulatory gene pathways is crucial for fully understanding apoptosis in human cells. In C. elegans, motorneuron apoptosis is regulated via the ces2→ces1→egl1 pathway. We propose that an analogous pathway exists in humans. It has been confirmed that the ces2 ortholog E4BP4 and the egl1 orthologs bim/puma are regulators in glucocorticoid (GC)-evoked apoptosis in human lymphoid leukemic CEM cells. We aim to determine whether E4BP4 regulates bim/puma in a manner similar to ces2's regulation of apoptosis via the down regulation of the ces1 ortholog slug/snail. In this study, E4BP4-dependence of slug/snail and bim/puma regulation by GCs was evaluated in CEM sister cell lines. Cells were treated with ethanol (control) or 1 μM Dexamethasone (a synthetic GC) for 24 h for evaluation of transcript and protein levels, and for 96 h for trypan blue exclusion-based viability assays. Relative mRNA expression and protein abundance of bim/puma and slug/snail were evaluated by qRT-PCR and Western analysis, respectively. Both bim/puma and slug/snail promoters were analyzed using promoter scanning software (Transcriptional Regulatory Element Database) to locate potential E4BP4 response elements. GC-evoked apoptosis in sensitive CEM-C7-14 cells correlated with E4BP4 upregulation, and GC-resistance in CEM-C1-15 cells could be overcome by stable ectopic expression of mouse E4BP4 (cell line CEM-C1-15-mE#3). Flow cytometric analysis of DNA content following propidium iodide labeling has confirmed the expected G1 arrest and accumulation of sub-G1 populations in C7-14 and C1-15-mE#3 cells. qRT-PCR and Western blotting results have confirmed differential bim expression; with C7-14 cells showing a significant two-fold increase in comparison to C1-15 cells. qRT-PCR results for puma, slug, and snail did not show significant differences in expression levels between C7-14, C1-15 and C1-15-mE#3 cells. Furthermore, the expected down regulation of slug/snail in the GC-sensitive C7-14 cells was not observed. Our data suggest that in CEM cell apoptosis, E4BP4-dependent downregulation of slug/snail is not an intermediate for bim upregulation. Unlike the ces2→ces1→egl1 pathway in C. elegans, E4BP4 may use alternative pathways (c-myc, c-jun, cyclin D3, etc.) or directly upregulate bim to evoke apoptosis in CEM cells. JAB is a recipient of a CSUN Graduate Equity Fellowship & a Graduate Fellowship for Outstanding Research Promise in Science & Mathematics from the CSUN College of S&M. Project funding by NIH grant (SC3 GM 081099-01) awarded to RDM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1028.