Glucocorticoid-mediated co-regulation of RCAN1-1, E4BP4 and BIM in human leukemia cells susceptible to apoptosis.

Glucocorticoids (GCs) are known to induce apoptosis of leukemia cells via gene regulatory changes affecting key pro-and anti-apoptotic genes. Three genes previously implicated in GC-evoked apoptosis in the CEM human T-cell leukemia model, RCAN1, E4BP4 and BIM, were studied in a panel of human lymphoid and myeloid leukemia cell lines. Of the two RCAN1 transcripts, the synthetic GC Dexamethasone (Dex) selectively upregulates RCAN1-1, but not RCAN1-4, in GC-susceptible Sup-B15, RS4;11, Kasumi-1 cells but not in GC-resistant Sup T1 and Loucy cells.

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.

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.

Quantitative polymerase chain reaction analysis by deconvolution of internal standard.

Background: Quantitative Polymerase Chain Reaction (qPCR) is a collection of methods for estimating the number of copies of a specific DNA template in a sample, but one that is not universally accepted because it can lead to highly inaccurate (albeit precise) results. The fundamental problem is that qPCR methods use mathematical models that explicitly or implicitly apply an estimate of amplification efficiency, the error of which is compounded in the analysis to unacceptable levels.

Results: We present a new method of qPCR analysis that is efficiency-independent and yields accurate and precise results in controlled experiments.

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.