Glucocorticoids in malignant lymphoid cells: Gene regulation and the minimum receptor fragment for lysis

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocortoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocortocoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half “Zn fingers” of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, proceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.     

Mechanism of the glucocorticoid regulation of growth of the androgen-sensitive prostate-derived R3327H-G8-A1 tumor cell line

The R3327H-G8-A1 cell line derived from the Dunning rat prostate adenocarcinoma contains both androgen and glucocorticoid receptors. Following steroid deprivation, androgens specifically increase the concentration of their receptors in these cells by approximately 2-fold within 6 h and 3-4-fold in 24 h. In the presence of potent glucocorticoids, androgen receptor augmentation is reduced by 40-50% in the first 6 h and completely inhibited during the subsequent 24 h. This event, which is specific for glucocorticoids, appears to be due to an inhibition of androgen receptor synthesis. Furthermore, glucocorticoids inhibit proliferation of these cells by inhibiting the release of growth factors and arresting them in the G0 or A state of the cell cycle. This inhibition can be overcome by addition of low concentrations of either epidermal growth factor or platelet-derived growth factor; however, the inhibitory effect of the glucocorticoid on androgen receptor augmentation is not released. These results suggest that glucocorticoids arrest cellular proliferation by altering the autoregulation of growth and that this event is not dependent upon inhibition of androgen receptor augmentation.     

Cyclic AMP-dependent protein kinase modulation of the glucocorticoid-induced cytolytic response in murine T-lymphoma cells

The antiproliferative effect of glucocorticoid hormones on lymphoid tissue serves as the basis for their use in chemotherapy of lymphomas and leukemias. The effectiveness of the steroid-mediated response is potentially contingent upon a variety of factors, including the cellular level of glucocorticoid receptors. This report demonstrates that differences in the expression of the glucocorticoid receptor gene can modulate steroid sensitivity of individuals within a population of lymphoma cells. We have also found that loss of cAMP-dependent protein kinase activity caused a measurable decrease of steroid sensitivity in the murine T-lymphoma WEHI-7 without producing a significant change in steroid binding capacity. However, the extent of this change in sensitivity was dependent upon the level of glucocorticoid receptor expression. Lymphoma cells containing few spare steroid receptors became significantly resistant to glucocorticoids through loss of cAMP-dependent kinase function. On the other hand, elevated levels of cAMP were found to cause an increase in glucocorticoid receptor mRNA concentrations. Thus, cAMP-dependent protein kinase activity has the potential to modulate a lymphoma cell's steroid sensitivity by affecting the level of glucocorticoid receptor expression as well as the receptor's efficiency in producing a cytolytic response.     

Evidence for platelet-activating factor receptors in several B lymphoblastoid cell lines.

Previous studies have shown that Raji, an Epstein-Barr virus (EBV)-immortalized Burkitt lymphoma B cell line, contains functional platelet-activating factor (PAF) receptors. Twelve other lymphoid cell lines, including Burkitt and non-Burkitt B cell lines, T cell lines, and a non B, non T cell line were tested for the presence of PAF receptors. Radioligand binding studies conducted at 4 degrees C revealed that six lymphoid cell lines of B cell origin (Raji, P3HR-1, BJAB, BJA/HR-1, Dakiki and PB-1) could specifically bind [3H]PAF. Treatment of four (Raji, P3HR-1, Dakiki and PB-1) of the above lymphoid cell lines with PAF resulted in an increase in free intracellular calcium, indicating that these specific PAF binding sites were functional PAF receptors. Other B cell lines (Daudi, B95-8, sfBT, CB-1), T cell lines (MOLT-4, CCRF-CEM) and a non B, non T cell line (NALM-6) had no PAF binding sites and showed no PAF-induced increase in intracellular calcium levels. These studies demonstrate evidence for the presence of PAF receptors on several B lymphocyte cell lines.     

A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line

Progesterone receptor-containing T47D human breast cancer cells are responsive to progestins but fail to respond to other steroid hormones, in particular dexamethasone, because they have no measurable levels of receptors for estrogens, androgens, or glucocorticoids. To quantitatively study dual responsiveness of the mouse mammary tumor virus (MMTV) promoter to progestins and glucocorticoids, we have stably transfected T47D cells with a glucocorticoid receptor (GR) expression vector. A cloned derivative (A1-2) was isolated that expresses a normal, full length GR, as assessed by steroid binding and Western immunoblot with a monoclonal anti-GR antibody. Moreover, GR is expressed at levels (80,000-100,000 molecules per cell) comparable to the high levels of endogenous progesterone receptor (200,000 molecules per cell). In A1-2 cells transiently transfected with an MMTV-chloramphenicol acetyl transferase reporter gene, induction by glucocorticoid was substantially greater (5-fold) than induction mediated by progestins. These results suggest that glucocorticoids may be the primary regulator of MMTV.     

Mechanisms of glucocorticoid function in human leukemic cells: analysis of receptor gene mutants of the activation-labile type using the covalent affinity ligand dexamethasone mesylate

In the cultured acute lymphoblastic leukemic (ALL) cell line, clones of sensitive cells are killed by receptor-occupying concentrations of glucocorticoids. In addition, several types of resistance have been identified. The types of resistance are r- (glucocorticoid binding site loss), ract/l (activation labile receptors) and r+ly- (defective lysis mechanism). The two types of receptor mutants have been examined for the presence and expression of the glucocorticoid receptor (GR) gene. Southern blot analysis, using a full-length cDNA probe for human GR, shows that the gene in both is grossly intact. Examination of the expression of the gene by Northern blots reveals the presence of normal, 7-kb message in both types of receptor mutants, though in amounts somewhat reduced from wild-type. This report focuses on the activation labile mutants. Since characterization of these mutants suggests that they can bind ligand but not retain it during activation, we hypothesized that they would respond normally to a ligand that could not be lost during activation. This seems to be the case. When the covalent affinity ligand dexamethasone mesylate, itself a partial glucocorticoid agonist/antagonist, is used, the ract/l cells are killed to an extent corresponding to that evoked by a sub-optimal concentration of the full agonist dexamethasone. We conclude: (1) that the ract/l receptors can function to kill cells if provided a ligand that they do not lose during activation; (2) that the partial agonist activity of dexamethasone mesylate for cell killing is not due to release of a small amount of free dexamethasone; (3) that the poor agonist activity of dexamethasone mesylate receptor complexes suggests that the role of steroid is strictly to participate in conversion of the receptor to its DNA binding form, after which presence of the steroid actually interferes with proper receptor action.     

Studies on arterial mineralocorticoid and glucocorticoid receptors: evidence for the translocation of steroid-cytoplasmic receptor complexes to cell nuclei

The high affinity binders for mineralocorticoids and glucocorticoids, previously reported by us as present in arterial cytosol have been further characterized. The results of this study demonstrate that these binders translocate under appropriate conditions to cell nuclei as complexes with mineralocorticoids and glucocorticoids, respectively. Thus, they exhibit a fundamental property of steroid receptors. This provides evidence for the presence in the arterial wall of a molecular mechanism(s) for the in-situ action of both mineralocorticoids and glucocorticoids.     

Glucocorticoid receptor-mediated expression of kallikrein 10 in human breast cancer cell lines

Using the breast cell lines MCF-10A, MDA-MB-468 and T-47D, we investigated the role of various glucocorticoids in regulating human kallikrein 10 expression. We found that increased concentrations of glucocorticoids decreased KLK10 expression in MCF-10A and increased KLK10 expression in MDA-MB-468 and T-47D cells. Stimulation of the cell lines using other steroid hormones did not yield any difference in KLK10 expression in MCF-10A and MDA-MB-468 cells, suggesting that regulation of KLK10 occurs primarily through glucocorticoids. However, T-47D cells expressed higher levels of KLK10 upon dihydrotestosterone stimulation. Blocking the glucocorticoid receptor (GR) demonstrated that the mechanisms of induction and repression are different in the three cell lines studied. Taken together, our results suggest an alternative mode of KLK10 regulation - by glucocorticoids via GR-dependent mechanisms.     

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells

Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells.Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid.Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed.Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.     

Binding studies of the antiglucocorticoid RU38486 in Daudi and Raji lymphoma cells

The activity of RU38486 has been studied in Burkitt's lymphoma cells which are Epstein-Barr virus (EBV) positive. The early antigens (EA) of the virus are induced by dexamethasone (DXM) in Daudi but not in Raji cells, whereas a growth factor (transforming growth factor-beta, TGF-beta) induces the EA in both cell lines. RU38486 blocks the EA induction obtained by DXM or by TGF-beta in either cell line. In order to understand the interaction of RU38486, we considered its binding to specific receptors. We first investigated the binding of the antagonist in whole cells at 22 degrees C. A number of specific binding sites higher for RU38486 than for DXM was found, suggesting that RU38486 may bind to the glucocorticoid receptor and also to other cellular structures which we called the antiglucocorticoid binding sites ("AGBS"). To support this hypothesis, competition experiments have been conducted between RU38486 and other steroid hormones (progesterone and testosterone) since it is known that RU38486 is also able to interact with their cognate receptors. Binding studies of RU38486 in vitro at 4 degrees C in the presence of cytosolic extracts from Daudi and Raji cells led to conclusions similar to those drawn from the whole cell experiments: more complexes were formed with RU38486 than with DXM. Finally, the steroid-receptor complexes were incubated with DNA-cellulose. Since the binding measured for RU38486 was higher than for DXM, we suspect that sites different from the classical glucocorticoid receptor sites are also able to interact with DNA. The blockage exerted by RU38486 on the EA induced by glucocorticoids or by non-steroidal molecules and the lack of responsiveness to glucocorticoids in Raji cells are discussed in the light of the present findings.     

Glucocorticoid effects on growth, and androgen receptor concentrations in DDT1MF-2 cell lines

The DDT1MF-2 smooth muscle tumor cell line contains receptors for and is differentially sensitive to androgens and glucocorticoids. Androgens stimulate and glucocorticoids inhibit growth. We now confirm that the latter involves the induction of a block in the G1 phase of the cell cycle. We have developed and characterized in vitro and in vivo a glucocorticoid resistant variant of this cell line, the DDT1MF-2-GR. Glucocorticoids specifically inhibit androgen induced androgen receptor augmentation in DDT1MF-2 cells, but not in the GR variant suggesting that growth inhibition is related to inhibition of androgen receptor augmentation. However, under optimal conditions for cell proliferation, when glucocorticoid inhibited growth is relieved by the exogenous addition of platelet derived growth factor, androgen receptor augmentation is still suppressed. Thus, androgen induced elevation in androgen receptor concentrations is not a prerequisite for cell proliferation. These results imply that in androgen responsive cells, although androgen stimulation of growth can be blocked by antagonism of androgen receptor mediated events, the antagonism can be bypassed by supplying the cells with exogenous growth factors. These results provoke speculation on how cells, which are dependent upon androgens for growth, become autonomous.