Abstract
BackgroundSynthetic GCs serve as therapeutic agents for some lymphoid leukemias because of their ability to induce transcriptional changes via the GC receptor (GR) and trigger apoptosis. Upregulation of the BH3-only member of Bcl-2 family proteins, Bim, has been shown to be essential for GC-evoked apoptosis of leukemic lymphoblasts. Using human T cell leukemic sister clones CEM-C7-14 and CEM-C1-15, we have previously shown that the bZIP transcriptional repressor, E4BP4, is preferentially upregulated by GCs in CEM-C7-14 cells that are susceptible to GC-evoked apoptosis, but not in refractory CEM-C1-15 cells. E4BP4 is an evolutionarily conserved member of the PAR family of bZIP transcription factors related to the C. elegans death specification gene ces2.ResultsMouse E4BP4 was ectopically expressed in CEM-C1-15 cells, resulting in sensitization to GC-evoked apoptosis in correlation with restoration of E4BP4 and Bim upregulation. shRNA mediated modest knockdown of E4BP4 in CEM-C7-14 cells resulted in concomitant reduction in Bim expression, although GC-evoked fold-induction and sensitivity to apoptosis was similar to parental cells.ConclusionData presented here suggest that GC-mediated upregulation of E4BP4 facilitates Bim upregulation and apoptosis of CEM cells. Since the Bim promoter does not contain any consensus GRE or EBPRE sequences, induction of Bim may be a secondary response.
Highlights
Synthetic GCs serve as therapeutic agents for some lymphoid leukemias because of their ability to induce transcriptional changes via the GC receptor (GR) and trigger apoptosis
We present evidence that E4BP4 plays a crucial role in GC-evoked apoptosis of CEM cells by enabling induction of Bim
The resistance in CEM-C-15 cells is thought to occur because of a blunted GR-dependent transcriptional response, several studies have focused on identifying key transcriptional changes that are unique to GCsensitive CEM cell clones [7,8,9]
Summary
Previous work has demonstrated the sensitivity and resistance of CEM-C7-14 and CEM-C1-15 cells, respectively, to GC-evoked cell death via apoptosis [7]. In Western blotting experiments using two different E4BP4-specific antibodies that recognize both the mouse and human homologs, it is clear that E4BP4 protein levels are not affected by Dex in CEM-C1-15 cells, but are induced in CEM-C7-14 and CEM-C1-15mE#3 cells (Figure 4D). To determine the effect of E4BP4 knockdown on Bim expression and sensitivity to GCs, CEM-C7-14 cells stably transfected with three different E4BP4-specific shRNA plasmids, ID1, ID2 and ID4, were created. Based on E4BP4 expression levels in parental CEM-C714 and CEM-C1-15 cells, it is apparent that rather than absolute abundance of E4BP4 and Bim transcripts, the change in expression in response to Dex determines sensitivity. Each of the shRNA transfected cell populations, and CEM-C7-14 cells were evaluated for their ability to upregulate E4BP4 and Bim expression in response to 1 μM Dex. all cell populations, including the knockdowns, showed a 4.5 to 7.5-fold upregulation of H. ID2 cells, which showed the maximum fold induction of E4BP4 and Bim expression, showed the greatest sensitivity to Dex-evoked cell death, confirming the correlation between E4BP4/Bim inducibility and susceptibility to cell death
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