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

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Abstract

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.

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Materials and methods

Reagents. Dexamethasone (Dex), ethylene glycol tetraacetic acid (EGTA), 2-bis(2-aminophenoxy)-ethane-N,N,N1,N1-tetraacetic acid-acetoxymethyl (BAPTA), and thapsigargin (TG) were purchased from EMD Biosciences (Madison, WI). Reagents for RT-PCR, including M-MLV reverse transcriptase, oligo(dT)15 primer, RNasin® Ribonuclease inhibitor, dNTP mix, and Taq DNA polymerase were purchased from Promega Life Sciences (Madison, WI). Calcium Green™-1 AM, and propidium iodide were purchased from Molecular

Sensitivity to GCs

As described in the literature [7], and shown in Figure 1s of Supplementary Materials, the CEM sister clones CEM-C7-14 and CEM-C1-15 are sensitive and resistant, respectively, to GC-evoked apoptotic cell death.

Correlation of increase in [Ca2+]i with apoptosis

Intracellular Ca2+ levels were monitored flow cytometrically using the fluorescent dye Calcium Green™-1 AM, a cell permeable dye, which upon binding to calcium, increases the intensity of fluorescence emission at 530 nm in proportion to the calcium concentration. Cells were simultaneously

Discussion

GC-evoked T-lymphocyte apoptosis has been studied extensively, yet the molecular pathway remains unclear. The process requires de novo RNA and protein synthesis, suggesting that gene regulatory changes are crucial for apoptosis induction [26]. GCs regulate a wide array of genes, reflecting their diverse actions on cell physiology. Because of the pleotropic effects of GCs, it has been difficult to correlate GC-evoked apoptosis with specific gene regulatory changes. Studies on individual genes,

Acknowledgments

This work was supported by a NIH MBRS-SCORE Pilot grant (S06-GM048680-10S1) awarded to RDM, the CSUN Office of Graduate Studies, Research and International Programs, and the CSUN College of Science and Mathematics. D.M. was supported through a NASA CSUN/JPL Pair Program fellowship for undergraduate students. We thank Dr. E.B. Thompson (UTMB, Galveston, TX) for kindly providing CEM-C7-14 and CEM-C1-15 cells. We thank the UCLA Flow Cytometry Core Facility for assistance with flow cytometric

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    Present address: Graduate Program in Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, CA 90095, USA.

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