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.