Glucocorticoids are commonly used in the first-line treatment of hematological malignancies, such as acute lymphoblastic leukemia, due to the ability of these steroids to activate pro-apoptotic pathways in human lymphocytes. The goal of the current study was to examine the gene expression and enzyme activity of the microsomal enzyme, 11-
Glucocorticoid (GC) therapy remains one of the first-line therapies in the treatment of a variety of leukemias, including acute lymphoblastic leukemia (ALL). Synthetic GCs, such as dexamethasone and prednisone, are effective in treating ALL due to their ability to induce cell cycle arrest and apoptosis.
The microsomal enzyme, 11-beta hydroxysteroid dehydrogenase type 2 (HSD2), catalyzes the conversion of biologically active 11-hydroxy glucocorticoids (i.e., cortisol) to their inactive 11-keto metabolites (i.e., cortisone). This activity allows for the tissue-specific conversion of cortisol to inactive cortisone, thereby decreasing the local concentrations of active GCs. Loss of the ability to inactivate cortisol via HSD2 enzyme activity has been implicated in the pathogenesis of a number of human diseases [
Despite the widespread use of GCs to treat leukemia, relatively little is known about the metabolism of GCs in human leukemic cells. The CEM-C7 cell line is glucocorticoid-sensitive subclone of a human leukemic cell line derived from a 4-year-old female patient with late stage ALL [
Glucocorticoid-sensitive CEM-C7 cells were grown in suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic/antimycotic solution, and 2 mM L-glutamine (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 95% air/5% CO2. Media were changed biweekly, and the cells were passed weekly, maintaining cell densities of
CEM-C7 cells were spotted and grown in serum-containing culture medium on chambered glass Lab-Tek microscope slides (Thermo Scientific, Rochester, NY) for 48 hours prior to immunostaining. Cells were fixed in formalin (10% formaldehyde in phosphate buffered saline) and immunostained as previously described using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and HSD2-specific antibody at a concentration of 200 ng/mL, or secondary antibody alone for negative controls [
Total RNA was isolated from CEM-C7 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s directions. The RNA was resuspended in ultrapure water and quantified by determining the absorbance at 260 nM. RNA integrity was monitored by examining the 28S and 18S rRNA bands after electrophoresis in agarose gels stained with ethidium bromide.
One microgram of total RNA was reverse transcribed using oligo-dT 18 mers as primers in a reaction volume of 30
Control and treated CEM-C7 cells grown to approximately 80% confluency in 100 mm culture plates were washed with phosphate buffered saline and then incubated in serum-free media containing 1,2-[3H]-cortisol (41 Ci/mmol, American Radiolabeled Chemicals, St. Louis, MO) at a final concentration of 1 nM for 24 hours at 37°C. Following incubation, the CEM-C7 cells were harvested by centrifugation for 10 minutes at 1,000 ×g. The labeled steroids were extracted from the media by adding 2 volumes of ethyl acetate and vortexing for 1 minute followed by centrifugation for 10 minutes at 600 ×g. The ethyl acetate phase was dried under N2 gas and the steroids resuspended in ethanol. The resuspended steroids were spotted onto silica-coated glass plates (silica gel G, Analtech, Newark, DE) and resolved in an equilibrated thin-layer chromatography (TLC) chamber using chloroform/ethanol (92 : 8) as the solvent. [3H]-Steroids were detected and quantified using a TLC plate reader (Bio Scan AR-2000, Bio Scan Inc., Washington, DC) capable of detecting tritium. HSD2 oxidase activity was assessed by calculating the conversion of [3H]-cortisol to [3H]-cortisone. Parallel assays were performed in flasks containing no cells to monitor for spontaneous interconversion between 11-hydroxy- and 11-dehydroforms of the steroids. Percent conversion values were normalized to protein content total protein homogenates from corresponding CEM-C7 cell pellets.
Total homogenate protein was isolated by homogenization of CEM-C7 cells, adult human liver tissue, or adult human kidney tissue (BioChain Institute, Hayward, CA) in lysis buffer (1 nM phenylmethylsulfonyl fluoride in PBS), followed by centrifugation (10 minutes at 600 ×g) and isolation of the supernatant. Protein concentration of the lysates was quantified using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). One hundred micrograms of homogenate protein from CEM-C7 cells or fifty micrograms of human kidney or liver homogenate protein was separated via one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions and subsequently transferred to nitrocellulose membrane. Immunoblotting was performed using a human HSD2-specific primary antibody (HUH23) at a concentration of 2
In order to assess the expression of the HSD1 and HSD2 genes, reverse transcription PCR (RT-PCR) was performed on total mRNA isolated from CEM-C7 cells using PCR primers specific for the HSD1 and HSD2 mRNAs. As shown in Figure
Expression of HSD2 in CEM-C7 cells. (a) mRNA for HSD2 was detected by RT-PCR analysis of total mRNA isolated from CEM-C7 cells. (b) Immunoreactive HSD2 protein was detected in homogenates of CEM-C7 cells, and human kidney was included as a positive control. (c) Cellular expression of HSD2 was also detected via immunohistochemistry.
In order to confirm the functional activity of the HSD2 gene in CEM-C7 cells, the metabolism of radioactively labeled cortisol was examined in cultured, intact cells. As shown in Figure
HSD2 enzyme activity in CEM-C7 cells and its regulation by Dex. (a) Intact CEM-C7 cells actively metabolized 3H-labeled cortisol to cortisone, indicative of HSD2-specific enzyme activity. Pretreatment of CEM-C7 cells with 100 nM Dex reduced HSD2-specific enzyme activity. (b) The Dex-mediated reduction in HSD2 activity was blocked by 2
In this paper, we have shown that a glucocorticoid-sensitive subclone of the CCRF-CEM cell line, CEM-C7, expresses the mRNA and protein for the GC metabolizing enzyme, HSD2. We have further shown that these cells also possess functional HSD2 enzyme activity, as demonstrated by the ability of these cells to oxidize biologically active cortisol to cortisone. In addition, it appears that the activity, and perhaps gene expression, of HSD2 is regulated by GR expressed in these cells. This is demonstrated by the finding that HSD2 enzyme activity is downregulated in cells that are pretreated for <15 hours and that this downregulation is completely blocked by the GR antagonist, RU-486. The effect of RU486 demonstrates that the regulation of HSD2 activity by Dex is a receptor-mediated response rather than a potential allosteric effect caused by direct interaction between the enzyme and the GR agonist (Dex).
As shown in Figure
In addition to observing the functional activity of the HSD2 enzyme in CEM-C7 cells, we also observed that the GR agonist, Dex, downregulates the amount of HSD2 enzyme activity present in these cells. For our studies, we chose to use Dex, since it is known to be a poor substrate for oxidative metabolism by HSD2 (thereby limiting its breakdown following administration) and also since Dex retains significant GR agonist activity in its 11-keto form [
To our knowledge, this paper represents the first report of HSD2 gene and enzyme activity expression in the CEM-C7 cell line. One recent study by Sai et al. demonstrated that the protein and mRNA expression of HSD2 was negligible in the GC-sensitive CCRF-CEM cell line but was more abundant in another human leukemic cell line that is GC resistant (MOLT4F cells) [
In summary, this paper presents the first data concerning the expression of the HSD2 gene in the glucocorticoid-sensitive CEM-C7 human leukemic cell line. In addition to confirming the expression of HSD2 mRNA, we have also demonstrated the expression of HSD2 protein, its cellular localization, and the functional activity of the HSD2 enzyme as evidenced by the conversion of cortisol to cortisone. We also demonstrate that the glucocorticoid receptor agonist, Dex, influences the activity of the HSD2 enzyme in these cells, likely in a GR-dependent manner as this downregulation is blocked by the GR antagonist, RU-486. This, and future work, is aimed at elucidating the potential role of glucocorticoid-metabolizing enzymes in human leukemic cells and how these cells metabolize different endogenous and synthetic glucocorticoid hormones.
The authors have no conflict of interests to disclose.
M. R. Garbrecht, the primary author, conducted experiments, experimental design, and data analysis. T. J. Schmidt also conducted experimental design, data analysis and assisted in paper preparation.
The authors wish to thank Dr. Zygmunt Krozowski for providing the HSD2 primary antibody (HUH23).