Correlation of glucocorticoid-mediated E4BP4 upregulation with altered expression of pro- and anti-apoptotic genes in CEM human lymphoblastic leukemia cells.

In Caenorhabditiselegans, motorneuron apoptosis is regulated via a ces-2-ces-1-egl-1 pathway. We tested whether human CEM lymphoblastic leukemia cells undergo apoptosis via an analogous pathway. We have previously shown that E4BP4, a ces-2 ortholog, mediates glucocorticoid (GC)-dependent upregulation of BIM, an egl-1 ortholog, in GC-sensitive CEM C7-14 cells and in CEM C1-15mE#3 cells, which are sensitized to GCs by ectopic expression of E4BP4. In the present study, we demonstrate that the human ces-1 orthologs, SLUG and SNAIL, are not significantly repressed in correlation with E4BP4 expression. Expression of E4BP4 homologs, the PAR family genes, especially HLF, encoding a known anti-apoptotic factor, was inverse to that of E4BP4 and BIM. Expression of pro- and anti-apoptotic genes in CEM cells was analyzed via an apoptosis PCR Array. We identified BIRC3 and BIM as genes whose expression paralleled that of E4BP4, while FASLG, TRAF4, BCL2A1, BCL2L1, BCL2L2 and CD40LG as genes whose expression was opposite to that of E4BP4.

E4BP4 facilitates glucocorticoid-evoked apoptosis of human leukemic CEM cells via upregulation of Bim.

BACKGROUND: 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. 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. RESULTS: Mouse 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. CONCLUSION: Data 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.

Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis.

BACKGROUND: Glucocorticoid hormones (GCs) induce apoptosis of leukemic T-cells by transcriptional regulation via the GC receptor, GR. In the human leukemic CEM cell culture model, RCAN1 has been identified as one of the genes that is specifically upregulated only in the GC-sensitive CEM C7-14 cells, but not in the GC-resistant CEM-C1-15 sister cells in correlation with GC-evoked apoptosis. RCAN1 gene encodes two major protein isoforms of the regulator of calcineurin (RCAN1), RCAN1-1 and RCAN1-4 via alternative splicing of exons 1 and 4 respectively, to exons 5-7. Studies reported here evaluated the differential regulation and function of the two transcripts and protein products of RCAN1 by the synthetic GC dexamethasone (Dex), and by modulators of calcium signaling. RESULTS: Dex selectively upregulates transcript specific for RCAN 1-1 in glucocorticoid (GC)-susceptible human leukemic CEM-C7-14 cells but not in GC-refractory CEM-C1-15 sister cells. Expression of the second major transcript, RCAN1-4, is upregulated by [Ca2+]i inducers, thapsigargin and A23187, but not by Dex, suggesting a mutually exclusive regulatory pathway for both RCAN1 transcripts. GC-mediated upregulation of RCAN1-1 transcript and RCAN1-1 protein was kinase dependent, and was blocked by staurosporine and the p38 MAP kinase inhibitor SB 202190. RCAN1-1 coimmunoprecipitates with calcineurin PP3C and Dex-mediated RCAN1-1 upregulation correlated with reduction in calcineurin PP3C activity. CONCLUSION: Data presented here suggest that GCs specifically upregulate RCAN1-1 transcript and protein while inducers of [Ca2+]i selectively upregulate RCAN1-4. GC-mediated increase in RCAN1-1 abundance and binding possibly inhibits calcineurin activity and modulates apoptosis in CEM-C7-14 cells.

Calcium-dependent upregulation of E4BP4 expression correlates with glucocorticoid-evoked apoptosis of human leukemic CEM cells.

Glucocorticoid (GC)-evoked apoptosis of T-lymphoid cells is preceded by increases in the intracellular Ca2+ concentration ([Ca2+]i), which may contribute to apoptosis. This report demonstrates that GC-mediated upregulation of the bZIP transcriptional repressor gene, E4BP4, is dependent on [Ca2+]i levels, and correlates with GC-evoked apoptosis of GC-sensitive CEM-C7-14 cells. Calcium chelators EGTA and BAPTA reduced [Ca2+]i levels and protected CEM-C7-14 cells from Dex-evoked E4BP4 upregulation as well as apoptosis. In the GC-resistant sister clone, CEM-C1-15, Dex treatment did not induce [Ca2+]i levels, E4BP4 expression or apoptosis, however, the calcium ionophore A23187 restored Dex-evoked E4BP4 upregulation and apoptosis. CEM-C7-14 cells were more sensitive to GC-independent increases in [Ca2+]i levels by thapsigargin, and a corresponding increase in E4BP4 expression and cell death, compared to CEM-C1-15 cells, suggesting a direct correlation between [Ca2+]i levels, E4BP4 expression, and apoptosis.