Rapid actions of glucocorticoidsPlasma membrane-resident glucocorticoid receptors in rodent lymphoma and human leukemia models
Introduction
The ability of glucocorticoids (GCs) to inhibit growth and cause involution of lymphoid tissues has been known for more than four decades [1], and is the basis for using these steroids in the treatment of leukemias and lymphomas. Despite many theories and much experimentation, we still do not fully understand the mechanisms by which GCs kill cells. However, most agree that the effects are mediated by glucocorticoid receptors (GRs). It is generally agreed that an absence, quantitative reduction, or defect in GR ensures lack of response to steroids, including GC-induced lymphocytolysis. However, high receptor numbers do not always predict steroid responsiveness [2], [3]. Therefore, as important as the intracellular GR (iGR) is for many cellular responses, it may not be the only mediator of GC-induced cytolysis.
GC-induced cell killing follows the classic pattern of programmed cell death (apoptosis), a process that has been described in developing or involuting tissues of many organisms. It is, therefore, appropriate to extrapolate possible lymphocytolytic mechanisms from those elucidated in other systems. While research in this area is currently advancing on many fronts, it seems clear that apoptosis occurs through a collaboration of membrane-initiated and gene expression mechanisms [4]. Mutations in proteins involved in either genomic or nongenomic actions can vitiate apoptotic responses. Therefore, GC-induced apoptosis is likely to depend on signaling through both pathways. Although nongenomic actions have been linked most dramatically to very rapid actions of steroids (such as membrane channel openings), the consequences of activating pre-existing proteins can also be relatively slow. For example, changes in the activity of ion pumps or enzymes of limited quantity will take some time to manifest themselves. The activation of both proteases and calcium-activated nucleases have been implicated in classical apoptotic responses [4].
Several investigators have pointed to the possibility that a subclass of steroid receptors, including GR, is localized in the plasma membrane [5], [6], [7], [8], [9], [10]. The classical biochemical binding assay has been used to demonstrate most of these receptors. The two most frequently used methods for measuring GC binding are the cytosolic/nuclear and whole-cell assays. That receptor levels are usually much higher when whole-cell assays are applied [11], [12] suggests that this assay system may measure both intracellular and plasma membrane-associated GR (mGR). However, due to the partitioning of steroids in the plasma membrane lipid environment, the scientific community has been reluctant to accept the findings generated from binding data. In all of these instances, the function of membrane binding is yet to be directly determined. We have employed an alternative immunological approach for the study of mGR.
Useful model systems for studying the relevance of mGR to lymphocytolysis include a number of neoplastic T-cell lines derived from both human [13] and mouse [14] that are sensitive to GC-induced lysis in vitro [15]. We have developed our own mGR-deficient and mGR-enriched cell lines from the S-49 mouse lymphoma and human CCRF-CEM acute lymphoblastic leukemia (ALL) cell models to test the relationship of mGR to GC-induced lymphocytolysis; this review will summarize these data. We will describe the identification of a GR associated with the plasma membrane, structure-function characterization studies, and comparative analysis of mGR and iGR. Evidence for mGR will be compared in the mouse and human systems. Then we will address the possible clinical relevance of mGR with its implications in mediating the therapeutic effects of GC in leukemic patients. Our findings in various wild-type and mutant mouse lymphoid cell lines will be summarized, along with our data from a severely mGR-deficient line.
Section snippets
Description of anti-GR antibodies used
For the rodent mGR, we employed the anti-GR monoclonal antibodies (Abs) BUGR-1 and -2 [16]. Because these monoclonals do not recognize the human GR, we produced a peptide-directed Ab to the hinge region of the human GR [17]. This reagent cross-reacts with rodent GR, and is, therefore, useful for the studies described here. In some studies, more highly purified forms of both reagents have been prepared by peptide affinity chromatography [18] of the antihuman Ab and by IgG affinity chromatography
Molecular size characterization of mGR by SDS–PAGE
Further verification of the immunoreactive membrane protein as a GR was accomplished by analysis of partially purified membrane preparations from mGR++ S-49 cells, and CCRF-CEM cells. GR was competitively affinity-labeled with the covalently binding ligand 3H-dexamethasone mesylate on whole cells [23] or in membrane vesicles purified on sucrose step gradients [17], [25].
Simultaneous Western and autoradiographic analyses (competitively affinity-labeled whole cells) of mGR and iGR from S-49 cells
Factors influencing the expression of mGR
We have recently investigated mGR regulation by the cell cycle. CCRF-CEM cells were synchronized employing three different procedures: double thymidine block, thymidine/colcemid block, and colcemid block [24]. Following release from synchrony, DNA distribution and mGR levels were examined at different time points. Fig. 4 shows representative histograms for the DNA distribution (right panel) and mGR measurements (left panel) obtained from CCRF-CEM mGR++ cells with the double thymidine block
Molecular etiology of mGR
We have recently identified 4 GR transcript splice variants (1A, 1B, 1D, and 1E) in S-49 cells [39]. All of them differ in the 5′UTR and splice onto the common exon 2, 13 nt upstream from the translational initiation site. That the expression of transcript 1A is highly correlated with the expression of mGR, suggests that mGR is encoded by this transcript [26]. Cloning and in vitro translation experiments suggest that transcript 1A is indeed the molecular progenitor of mGR (manuscript
The correlation between lysis competence and the presence of mGR
We have published several papers correlating the presence of mGR with the apoptotic sensitivity of lymphocytes to the killing effects of GCs [20], [21], [22], [23], [24], [25]. Six lines of evidences can be provided to support this observation:
First, S-49 cells specifically enriched for mGR are more efficiently killed by GC treatment, compared to mGR− cells, Fig. 5A. The kinetics of cell death are of the first order for the mGR-enriched cells, with >98% lysed by the 4th day of dexamethasone
Clinical implications of mGR
Glucocorticoids are one of the most classically utilized drugs in the clinical management of lymphoproliferative diseases (reviewed in [24]). Despite the fact that we still do not understand the mechanisms by which GCs kill cells, there remains every reason to suppose that the effects are mediated by GRs. High numbers of lymphoid cell receptor sites have, for the most part, been correlated with good clinical response to GC treatment [1]; these correlations are strongest for childhood ALL and
Summary and overall conclusions
The identity of the mGR protein as a unique form, versus a modified form of the iGR, has been a point of major contention. Our studies show that mGR is more similar to, than different from, iGR. Differences include cellular localization, molecular size, and a slightly altered ability of other steroids to compete for glucocorticoid binding. However, similarities are numerous. Both mGR and iGR bind the same class of steroid, have epitope recognition for 3 anti-GR Abs, bind GRE DNA, have many
Acknowledgments
Other S-49 cell clones, termed the nuclear translocation reduced S-49.22r (NT−) and nuclear translocation increased S-49.143r (NTi) were provided by Dr. R. Miesfeld [44], while wild-type (W7MG1), dexamethasone-resistant (W7M326.4), and receptorless (ADR6.M189) lines, all derivatives of WEHI-7 cells, were obtained from Dr. M. Stallcup [45]. Dr. E. A. Thompson provided the P1798 cell lines [46]. We thank Dr. D. Konkel for critical review of our manuscript. This work was supported by grants from
References (47)
- et al.
Analysis of glucocorticoid receptor activation by high resolution two-dimensional gel electrophoresis of affinity-labeled receptor
J Biol Chem
(1986) - et al.
Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486
J Steroid Biochem
(1985) - et al.
Glucocorticoid uptake into human placental membrane vesicles
J Biol Chem
(1979) - et al.
Preparation of adsorbents for biospecific affinity chromatography. I. Attachment of group-containing ligands to insoluble polymers by means of bifunctional oxiranes
J Chromatography
(1974) - et al.
Studies on the arrangement of glucocorticoid receptors in the plasma membrane of S-49 lymphoma cells
Steroids
(1991) - et al.
Size and steroid-binding characterization of membrane-associated glucocorticoid receptor in S-49 lymphoma cells
Steroids
(1991) - et al.
The interaction of steroids with Rana pipiens oocytes in the induction of maturation
Dev Biol
(1971) - et al.
Phosphotryptic peptide analysis of human progesterone receptors. New phosphorylated sites formed in nuclei after hormone treatment
J Biol Chem
(1989) - et al.
Molecular genetic analysis of glucocorticoid and mineralocorticoid signaling in development and physiological processes
Steroids
(1996) - et al.
High glucocorticoid receptor content of leukemic blasts is a favorable prognostic factor in childhood acute lymphoblastic leukemia
Blood
(1993)