Abstract
Glucocorticoid (GC) is a major therapeutic agent for the treatment of leukemia because of its ability to induce apoptosis in lymphoid cells. The mechanism causing apoptosis, however, is still controversial. Since the glucocorticoid receptor is a transcription factor, some of its target genes are expected to be implicated in apoptosis. In this study, using a GC-sensitive human pre-B leukemia cell line, Nalm-6, the FK506 binding protein 51 (FKBP5) and regulator of calcineurin 1 (RCAN1) genes were disrupted by homologous recombination, since the expression of both is up-regulated by GC in GC-sensitive but not in GC-resistant leukemic cell lines. While the disruption of FKBP5 had a marginal effect on GC-induced apoptosis, that of RCAN1 resulted in marked resistance to GC. In addition, overexpression of RCAN1 rendered cells more sensitive to DEX. In RCAN1-disrupted cells, levels of some pro-apoptotic and anti-apoptotic Bcl-2 family proteins were decreased and increased, respectively. Finally, phosphorylation of cAMP-response element binding protein (CREB) and up-regulation of CREB target genes by GC were inhibited by RCAN1 disruption, and treatment with a cAMP-inducing agent, forskolin, restored the sensitivity to GC in RCAN1-disrupted Nalm-6 cells. These findings suggest that up-regulation of RCAN1 expression followed by activation of the CREB pathway is required in GC-induced apoptosis.
Highlights
Glucocorticoids (GCs) have a wide variety of pharmacological effects such as immunosuppression, anti-allergy, and anti-inflammation, and are used as chemotherapeutic agents for various leukemias, lymphomas, and multiple myelomas because of their ability to induce apoptosis and cell cycle arrest in lymphoid cells [1,2]
To determine whether the disruption alters the sensitivity to GC, we investigated the time course of cell death of wild-type and FKBP52/2 cells treated with 1 mM DEX
The percentage of annexin V-positive apoptotic cells among wild-type Nalm-6 cells started to increase at 24 hours after the DEX treatment and 40% of the cells were positive at 48 hours, while 63% of FKBP52/2 cells were apoptotic at this time point (Figure 1D)
Summary
Glucocorticoids (GCs) have a wide variety of pharmacological effects such as immunosuppression, anti-allergy, and anti-inflammation, and are used as chemotherapeutic agents for various leukemias, lymphomas, and multiple myelomas because of their ability to induce apoptosis and cell cycle arrest in lymphoid cells [1,2]. GCs diffuse through the cell membrane into the cytoplasm and bind to the intracellular glucocorticoid receptor (GR) to exert their effects on target cells. GR interacts with other transcription factors such as AP-1, STAT-5, and NF-kB [6,7,8]. These interactions affect transcription levels of the target genes by modifying the actions of the GR partners
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