Substitution of arginine for cysteine 643 of the glucocorticoid receptor reduces its steroid-binding affinity and transcriptional activity
Introduction
Glucocorticoid receptor (GR) is a transcription factor activated by glucocorticoids. GR exists as a cytosolic protein associated with several heat-shock proteins and other proteins [1]. Glucocorticoids bind the cytosolic GRs, which leads to the nuclear translocation of GR [2]. The induction or suppression of the transcription of target genes varies depending on tissues or cell types, thus accounting for the diverse and sometimes opposite physiological effects of glucocorticoids on different tissues. It is well known that GR is involved in the glucocorticoid-induced apoptosis in lymphocytes [3], [4].
Glucocorticoids are one of the agents used for the treatment of childhood acute lymphoblastic leukemia, and included in standard remission induction regimens [5], [6]. The previous studies showed that human leukemic CEM-C1 cells are resistant to glucocorticoid-induced apoptosis and have a heterozygous mutation (L753F) in the ligand-binding domain (LBD) of the GR gene [7], [8], [9], [10]. In those cells, glucocorticoid-binding affinity and the transcriptional activation were significantly reduced. Therefore, the mutation L753F is considered to be responsible for the glucocorticoid resistance in the CEM-C1 cells.
In this study, we sequenced the coding regions of the GR gene in ten individual Japanese leukemic cell lines to find another mutation influencing its transcriptional activity.
Section snippets
DNA source and sequencing of GR gene
Established leukemic cell lines derived from the ten different Japanese individuals (JCRB0071, 0086, 0091, 0092, 0094, 0104, 0105, 0112, 0114 and 0122) were obtained from the Health Science Research Resources Bank (Osaka, Japan) or from the Japanese Collection of Research Bioresources, National Institute of Health Sciences (JCRB, Tokyo, Japan). DNA extraction was carried out using the Blood and Cell Culture DNA Kit (Qiagen, Hilden, Germany).
The primers for the polymerase chain reaction (PCR)
A mutation C643R in exon 7 in GR gene
We sequenced the coding regions of GR gene in ten Japanese leukemic cells, but could not find the previously reported L753F mutation. However, we detected a heterozygous mutation t5618c (the numbering is based on the reference sequence, GenBank Accession Number: AC004782) (surrounding sequence; tacgaccaa T/C gtaaacacat), which changes cysteine 643 to arginine (exon 7) of GR. The mutation (C643R) was detected in the cell line P30/OHK (Health Science Research Resources Bank Accession Number:
Discussion
Glucocorticoid resistance is a major problem in the treatment of acute lymphatic leukemia, and some reports about underlying mechanisms have been shown, yet very little is known. The mutation L753F was detected in the human steroid-resistant leukemic CEM-C1 and ICR27TK.3 cells, and considered to cause their resistance [10]. In this paper, in order to investigate whether the mutation L753F exists or not in other leukemic cells, we sequenced the exon 9 of GR gene in ten Japanese individual
Acknowledgements
This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ-6) of the Organization for Pharmaceutical Safety and Research of Japan. The authors would like to thank Hideto Jinno, Mayumi Saeki and Akiko Soyama for technical help in the DNA preparation.
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