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. Author manuscript; available in PMC: 2009 Oct 27.
Published in final edited form as: J Steroid Biochem Mol Biol. 2000 Dec 15;75(2-3):91–99. doi: 10.1016/s0960-0760(00)00161-8

The delayed induction of c-jun in apoptotic human leukemic lymphoblasts is primarily transcriptional

Feng Zhou 1,1, Rheem D Medh 1, Weiping Zhang 1, Naseem H Ansari 1, E Brad Thompson 1,*
PMCID: PMC2768356  NIHMSID: NIHMS147929  PMID: 11226825

Abstract

Because of their ability to induce lymphoid cell apoptosis, glucocorticoids have been used for decades to treat certain human leukemias and lymphomas. Studies presented in this paper complement our previous work demonstrating that sustained induction of the proto-oncogene c-jun plays a crucial role in the glucocorticoid-induced apoptotic pathway in CEM cells, human leukemic lymphoblasts. Results from measurements of c-jun mRNA half-life with RNase protection assays and of transcription by nuclear run-on assays indicate that, in the dexamethasone-sensitive cloned CEM-C7 cells, c-jun is induced at the transcriptional level. Consideration of time-course, however, suggested that this might be a secondary or possibly a delayed primary response. Use of cycloheximide to block protein synthesis strongly induced c-jun mRNA, suggesting that there had been relief from a labile protein repressor of transcription. Comparing the level of induction by cycloheximide with that of dexamethasone indicated that the two did not induce by an identical mechanism. The high induction by cycloheximide obscured simple interpretation of elevated c-jun mRNA levels after concomitant administration of cycloheximide and dexamethasone. This was resolved by nuclear run-on experiments, which showed that the dexamethasone induction of c-jun mRNA in this system does require protein synthesis.

Keywords: Glucocorticoids, c-jun Gene regulation, Apoptosis, Lymphoid, CEM cells

1. Introduction

The sustained induction of c-jun has been implicated as a causative signal in various apoptotic pathways [1–5]. We previously reported data suggesting that c-Jun, the protein coded by the c-jun proto-oncogene, is critical to the glucocorticoid-evoked apoptosis of a human leukemia lymphoblast line [6]. The evidence showed that: (1) c-jun mRNA and c-Jun protein, but not c-Fos, JunB or JunD, are induced by dexamethasone (Dex) in CEM lines that are sensitive to glucocorticoid-dependent apoptosis; (2) c-jun induction occurs before overt apoptosis; (3) among CEM clones, a correlation exists between high-level expression of c-jun and glucocorticoid-evoked apoptosis; and (4) anti-sense c-jun blocks both Dex induction of c-jun mRNA and apoptosis.

The proto-oncogene c-jun is an immediate early response gene. Thus, when resting cells are stimulated to grow, a transient burst of c-jun transcription occurs shortly after addition of the stimulus. In cycling cells, c-jun is expressed briefly during a part of the G1 phase of cell cycle. The effects of c-jun transcription are manifested through its coded protein c-Jun, part of the AP-1 transcription complex family of proteins [7]. Inappropriate, often prolonged expression of c-Jun has been found correlated with apoptosis and, in several cases, blocking this expression has been protective [3,6].

When glucocorticoids are added to cultures of cloned CEM-C7 cells, several early molecular events occur. These include rapid downregulation of c-myc [8,9], followed in a few hours by induction of c-jun mRNA and protein [6]. This continues to increase to a maximum, plateauing at around 24 h. About that time, cells begin to show several biochemical and gross morphologic manifestations of apoptosis [10], which increase thereafter, until most cells are dead. As long as agonist steroid is present and cells are viable, the c-Jun levels remain high. The pattern of delayed induction displayed by c-jun is atypical for a ‘primary’ induced gene. It is known that genes simply and directly induced by glucocorticoids show an increase in transcription in a short time after addition of steroid (see Ref. [11] and references cited therein). Activation of the glucocorticoid receptor (GR) and its nuclear translocation take only minutes [12]. The delay of ≥6 h before accumulation of mRNA over basal levels, as is seen with c-jun induction [6], suggests either a very large initial pool of mRNA (so it is a while before total c-jun mRNA can be seen to increase), very weak induction, delayed post-transcriptional regulation, or ‘secondary’ induction. In secondary induction, either a necessary factor must be induced or a repressor destroyed before the mRNA measured can increase. The present studies were designed to distinguish between these general mechanisms, specifically to test whether induction of c-jun was by transcriptional or post-transcriptional mechanisms and, if transcriptional, whether by the primary or secondary mode.

2. Materials and methods

2.1. Cell lines

The glucocorticoid-sensitive cell clone CEM-C7 was derived from CCRF-CEM, obtained from a female patient with acute lymphoblastic leukemia [13]. ICR-27 cells are a clonal derivative of CEM-C7 cells, obtained after mutagenesis and selection for resistance to high-dose Dex and have a defective GR [14,15].

2.2. Protein extraction and Western blot

Cells were washed with phosphate-buffered saline (PBS) (4°C) and resuspended in a protease inhibitor cocktail (50 mM Tris (pH 7.4), 0.6 M NaCl, 0.4 mM AEBSF, 80 µM leupeptin). Cells were lysed by performing three cycles of freezing for 5 min in an alcohol/dry ice mixture followed by thawing for 30 min in a 4°C ice water bath. The cellular lysates were centrifuged at 30,000 r.p.m. for 30 min, at 4°C using a Beckman TL-100 ultracentrifuge (Beckman Instruments, Palo Alto, CA). The supernatants were removed to fresh tubes. Protein concentration of the extract was then estimated using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Whole cell extracts containing 50 µg protein were electrophoresed in 12% polyacrylamide minigels (Bio-Rad) containing sodium dodecyl sulfate (SDS) and transferred to nylon membranes (Bio-Rad) using a semidry electroblotter (Integrated Separation Systems, Hyde Park, MA). After incubation in 10% dry milk powder in PBS, the membranes were incubated at 4°C in PBS containing a solution of 5% powdered milk with the c-Jun polyclonal antibody source. After washing with PBS, the membranes were incubated for 5 min in the horeradish peroxide substrate (Pierce Chemical Company, Rock-ford, IL), and developed on film using various times of exposure to assure that evaluations for quantification of the signals were within the linear response range. The densitometric analysis of c-Jun protein was performed using a Lynx densitometer (Applied Imaging, Santa Cruz, CA).

2.3. Nuclei isolation and nuclear run-on assay

Nuclei from CEM-C7 cells were isolated according to published methods (Current Protocols in Molecular Biology, Supplement 26, 1994). Briefly, cells were cultured as already described and resuspended in an iso-osmotic buffer (0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM ethylenediamine tetraacetic acid (EDTA), 10 mM Tris–HCl (pH 8.0), 1 mM dithiothreitol (DTT), 0.5% (v/v) Nonidet P-40). The nuclei were obtained by layering the lysed cell suspension on a sucrose cushion (2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris–HCl (pH 8.0), 1 mM DTT) and subjecting the samples to ultracentrifugation at 19,000 r.p.m. at 4°C in a SW 50.1 rotor, for 47 min with slow acceleration and deceleration. Nuclei were immediately frozen and stored at −70°C until use. Nuclear run-on transcription assays were carried out according to Mahajan and Thompson [16]. Briefly, in a final volume of 400 µl, 200 µl nuclear suspension was mixed with the nuclear run-on transcription mixture (0.5 M Tris–HCl (pH 7.8), 0.25 M NaCl, 1.75 M ammonium sulfate, 10 mM EDTA), 0.2 M MnCl2, 20 mg/ml heparin, a mixture of adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate (20 mM stock, 1 mM final concentration), and labeled uridine triphosphate (600 Ci/mmol, 150 µCi/reaction). The reaction mixture was incubated at 30°C for 45 min followed by the addition of yeast tRNA (1 mg/ml), 20 mM Tris–HCl (pH 7.5), 10 mM CaCl2, and a solution of DNase I with proteinase K (v/v, 1:1). The samples were incubated for 30 min at 37°C, RNA was extracted, and to the aqueous phase, 20% TCA was added. After incubating on ice, the samples were centrifuged for 15 min at 10,000 × g, and rinsed twice with ice-cold 5% TCA. After dissolving the pellet, RNA was precipitated overnight, dissolved in RNase-free water, and aliquots were added to filters and counted in a scintillation counter. The labeled RNA was used for hybridization as follows. The linearized double-stranded c-jun and actin plasmids vector or a single-strand plasmid were denatured and applied onto nitrocellulose using a slot blot apparatus. The filter was baked for 2 h at 80°C, prehybridized in the hybridization solution (0.01 M TES (pH 7.4), 0.2% SDS, 0.01 M EDTA, 0.3 M NaCl, 1 × Denhardt’s, 100 µg/ml yeast tRNA, 100 µg/ml poly A) at 65°C for 2–6 h, and hybridized in the same hybridization solution but with added labeled RNA ((1–5) × 107 cpm/ml) for 30–40 h at 65°C. The filters were washed with 2 × SSC at 65°C for 1 h followed by a 30-min wash at 37°C with 2 × SSC and 10 µg/ml boiled RNase A. The filters were then exposed to a phosphorimager screen and the relative amounts of c-jun and β-actin nuclear run-on transcripts were quantitated by using the MD IMAGEQUANT software (version 3.3; Molecular Dynamics, Sunnyvale, CA). Normalization was carried out using β-actin as the internal control.

2.4. Determination of caspase activity

Caspase activity was measured as described by Sarin et al. [17] with slight modifications. CEM-C7 cells treated with or without 100 nM Dex were harvested and 2.5 × 105 cells were lysed in 50 µl buffer containing 50 mM HEPES (pH 7.5), 10% sucrose, 0.1% Triton X-100 and 10 µM DTT. Substrates for measurement of caspase activity were obtained from Enzyme Systems Products (Livermore, CA). Caspase 1, 2, 3 and 8 were measured using substrates Z-YVAD-AFC, Z-VDVAD-AFC, Z-DEVD-AFC and Z-IETD-AFC, respectively. The cell lysate (100 µl) was mixed with 50 µM appropriate fluorogenic substrate, incubated at room temperature for 1 h, diluted to 1 ml with PBS and the fluorescence of the AFC [7-amido-4-trifluoromethylcoumarin] released was measured using the F-4500 fluorescence spectrophotometer (Hitachi Instruments, San Jose, CA).

2.5. RNA purification and RNase protection assays

Cells were collected, quickly frozen and stored at −70°C until all samples were ready for analysis. Total RNA was isolated using the TRIzol reagent (Gibco/BRL Life Technologies, Grand Island, NY) according to the method provided by the manufacturer. A plasmid pTRI-c-jun containing 340 base pairs (bp) c-jun cDNA was constructed. 32P-labeled antisense c-jun RNA probe was synthesized by T7 promoter-dependent RNA polymerase according to standard in vitro transcription protocols (Ambion, Austin, TX), and purified by gel electrophoresis. RNase protection assay was carried out with a kit from Ambion. Basically, RNA samples were mixed with the labeled probe. The mixtures were added with 5 M NH4OAc and 2.5 volumes of EtOH and kept at −20°C for 15 min, then pelleted by centrifugation. The pellets were resuspended in hybridization buffer, incubated at 42–45°C overnight, digested by RNase, and precipitated. The pellets were resuspended in gel loading buffer, denatured and subjected to electrophoresis. The gel was dried and exposed to a phosphorimager screen. Image bands were quantified by the MD IMAGEQUANT software.

3. Results

3.1. Induction of c-jun is receptor dependent

Since glucocorticoid activation of its receptor and subsequent nuclear translocation are rapid processes, delay in the induction of c-jun could be due to some non-traditional, receptor-independent mechanism. Previously, we showed that c-jun induction occurred at Dex concentrations consistent with those required to occupy the GR. In this paper, we have tested whether these receptors are required for the induction. ICR-27 cells, a receptor-defective subclone of CEM-C7, were employed because their failure to respond is due simply to lack of wild-type receptor. Their Dex resistance is due to the deletion of the normal GR allele [15]. Restoring GR to these cells by complementation or transfecting it via an expression plasmid restores their apoptotic response to Dex [11,18]. ICR-27 cells were therefore treated with 1 µM Dex or ethanol vehicle for up to 48 h, during which time they grew continuously. At intervals, samples were taken for immunochemical analysis of c-Jun. No induction was seen at any time. An example of one experiment is shown in Fig. 1. Clearly, Jun levels were not increased in ICR-27 cells; if anything, they were slightly reduced and were constant at all time points following Dex treatment. Control CEM-C7 cells treated in parallel showed the typical induction.

Fig. 1.

Fig. 1

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Dex did not induce c-Jun in cells lacking functional GR, but did in cells with functional GR. ICR-27 (upper panel) or CEM-C7 (lower panel) cells were treated with 1 µM Dex. At 0, 12, 24 and 48 h, cell samples were extracted for immunoblot analysis of c-Jun levels.

3.2. The expression of c-jun is regulated by Dex at the transcriptional level

To determine whether c-jun induction occurs at a transcriptional or post-transcriptional level, cell nuclei were isolated from CEM-C7 cells at 0, 6, 12, 24 h after addition of 1 µM Dex to the cultures. Nuclear run-on experiments were performed to test for induction of transcription. The results indicate that c-jun transcription increased beginning at 6 h and continued to rise thereafter, reaching an average maximum of 2.6-fold at 24 h (Fig. 2). In no experiment was evidence of transcription induction seen earlier than 6 h.

Fig. 2.

Fig. 2

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Dex induced c-jun mRNA at the transcriptional level. CEM-C7 cells were treated with Dex for 0, 3, 6, 12, or 24 h. Nuclei isolated from cells sampled at each time point were used to assay for c-jun transcription, as described in Section 2. Data from one of three such experiments are shown. (A) Slot blots of the duplicate samples; (B) line graph of the data quantified by image analysis. The c-jun bands from (A) at each time point were quantified, averaged, and normalized to the β-actin bands for the respective time points. The experiment was repeated three times (except the 12 h time point, which was tested in four experiments) with similar results.

3.3. Capase activity increases after c-jun induction

Our previous work [6] showed that c-jun induction begins 6–12 h after Dex is added to CEM-C7 cells. In this paper, we confirm this result and show that this induction is transcriptional (Fig. 2). Overt apoptosis begins later, after about 24 h. To determine the chronological relationship between c-jun induction and the known predominant effector of apoptosis, the caspases, we assayed for changes in activity of caspases 1, 2, 3 and 8. The results showed (Fig. 3) that, whereas there was no change in activity of caspase 1 or 8, caspases 2 and 3 increased strongly between 24 and 36 h. These increases in activity were GR dependent, since they were not observed in the GR-deficient ICR-27 cells (data not shown).

Fig. 3.

Fig. 3

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Time course of Dex-evoked caspase activation in CEM-C7 cells. CEM-C7 cells were treated with 100 nM Dex for the indicated times; after which cells were harvested and lysed as described in the Section 2. The activities of caspases 1, 2, 3 and 8 were determined fluorometrically, using specific AFC substrates for each. The data shown are averages of duplicate determinations from a representative experiment. In five experiments, a similar time course of caspase 2 and 3 activation was observed.

3.4. Dex does not affect the stability of c-jun mRNA

Measurements of c-jun mRNA pools were performed to determine the half-life of the message after the addition of 1 µM Dex or ethanol (EtOH, control) for 6, 12, or 24 h. Fig. 4A–D shows the results from an experiment at the 12 and 24 h time points. The half-life of c-jun was identical in control and Dex-treated cells, although of course the absolute level of c-jun mRNA was much higher in the Dex-treated cells. The half-life of c-jun also showed no change after treatment of the cells with Dex 1 µM for 6 h (data not shown). In all cases, c-jun mRNA had a half-life of approximately 0.7 h.

Fig. 4.

Fig. 4

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Dex treatment did not stabilize c-jun mRNA in CEM-C7 cells. CEM-C7 cells were incubated with EtOH or Dex for 12 h (A, B), or 24 h (C, D). Actinomycin D (AD) was added to cells to a final concentration of 3 µM. Cells were collected at time intervals shown thereafter. The mRNA for c-jun in extracts containing total cellular RNA was estimated by RNase protection assay, performed as described in Section 2. The specific protected bands of c-jun from (A) and (C) were quantified and normalized to the corresponding β-actin bands. Data were plotted on a semi-log scale in (B) and (D). The same results were obtained in two independent experiments.

3.5. Levels of c-jun mRNA were increased by cycloheximide

A classic test for secondary induction is to block protein synthesis while inducer is added; then to follow the relevant mRNA [19]. Hence, to try to determine whether de novo synthesis of protein was required for the Dex induction of c-jun, 10 µg/ml cycloheximide (CHX) was added to the cultures to inhibit protein synthesis. However, CHX itself induced c-jun mRNA levels strongly. Continuous incubation with CHX alone for 6 or 12 h resulted in an eight- to tenfold increase in c-jun mRNA over the ethanol control-treated cells (Fig. 5A). This suggested that the CHX treatment had removed a repressor of c-jun. Perhaps the Dex induction occurred by removal of the same repressor. Consideration of the timing and quantity of c-jun mRNA pool induction suggested that Dex and CHX were not operating on the same repressor, at least not with equal efficiency. Fig. 5B presents data comparing the pool size change of c-jun mRNA after treatment with CHX alone (the same data as in Fig. 5A), Dex alone, or both together for 12 h. Obviously, the increase in c-jun mRNA was much greater after CHX treatment, and this large induction obscured any effect that blocking protein synthesis might have on the Dex induction process.

Fig. 5.

Fig. 5

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CHX increased c-jun mRNA to a higher level than did Dex. Total RNA was extracted from CEM-C7 cells, and the RNase protection assay was used to determine the c-jun mRNA levels. (A) Cells were treated with EtOH or CHX for 12 h, or CHX for 6 h; (B) cells were treated with EtOH, Dex, Dex + CHX and CHX for 12 h; (C) cells were incubated with ethanol for 12 h, Dex for 6 h, Dex for 12 h with CHX present for the initial 6 h (Dex12 + CHX6a), Dex for 12 h, and EtOH for 12 h with CHX for initial 6 h (CHX6a); (D) cells were incubated with EtOH for 12 h, Dex for 12 h, Dex for 24 h with CHX present for the initial 12 h (Dex24 + CHX12a), Dex for 24 h, and CHX for initial 12 h (CHX12a). All experiments were repeated with essentially the same results.

As quickly as CHX induces, so does its removal allow the c-jun mRNA to return to basal levels. Exposing cells to CHX for 6 or 12 h, followed by washes to remove the inhibitor, and reincubation for an equal time caused the mRNA to fall substantially towards basal levels (Fig. 5C,D). However, if CHX was present during the first 6 of 12 or 12 of 24 h while Dex was continuously present for the entire period, the final c-jun mRNA levels were as high as if Dex alone had been present for the entire 12 or 24 h (compare Fig. 5C,D). Two possibilities suggest themselves: either protein synthesis was not required for the Dex induction, and the superimposed CHX effect came and went unrelated to Dex induction; or the treatment with CHX together with Dex did in fact block induction by Dex but left the c-jun gene in a state that subsequently allowed Dex to induce it to a greater extent than would have been reached in the length of time available without blocking protein synthesis. Use of nuclear run-on assays permitted a more direct test of the need for prior protein synthesis for the Dex induction to occur.

3.6. Dex induction of c-jun transcription does require newly synthesized protein

Since CHX elevated c-jun mRNA to higher levels than that induced by Dex, making interpretation of its effects on the Dex-dependent induction by assays of mRNA pools difficult, nuclear run-on experiments were employed to address whether de novo protein synthesis was required. CEM-C7 cells were treated with EtOH, Dex, Dex + CHX, or CHX for 16 h, and their nuclei prepared for nuclear run-on assay. Fig. 6 shows that the level of new c-jun transcripts initiated after treatment with Dex for 16 h in the presence of CHX was significantly less than the level induced by Dex alone for the same period of time. This implied that CHX alters the effect of Dex on the expression of c-jun, and that de novo protein synthesis was required for the transcriptional induction of c-jun by Dex. We noted that Dex + CHX slightly increased the synthesis of c-jun mRNA. However, that increase was significantly less than that induced by Dex alone and might be due to the effect of CHX itself, which elevated c-jun synthesis 2.2-fold. Thus, a significant part of the Dex-dependent transcription induction of c-jun was blocked by CHX.

Fig. 6.

Fig. 6

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De novo protein synthesis was required for the induction of c-jun with Dex in CEM-C7 cells. Nuclei were isolated and the nuclear run-on assay was used to determine the newly synthesized c-jun mRNA levels. Single-strand DNA of a plasmid with c-jun in an antisense orientation was used to determine c-jun expression. A plasmid-containing actin was used as internal control and a vector without a c-jun insert was used for a negative control. (A) CEM-C7 cells were treated with EtOH, Dex, Dex + CHX or CHX alone for 16 h. Three lanes show triplicates. (B) The bands in (A) were quantified. Data from triplicates were averaged and subtracted by data from vector, and then normalized by data from actin. The data for EtOH treated were set as one-fold. The bars represent the range between two independent experiments.

4. Discussion

Products of the nuclear proto-oncogenes jun and fos play a major role during cell growth, differentiation and development [20]. Induction of c-Jun has been implicated as a mediator of lymphoid cell apoptosis evoked by various stimuli, including ceramide, stress, bufalin and corticosteroids [21–23]. Antisense c-jun or circumin, an inhibitor of AP-1 activity, was able to inhibit various instances of apoptosis [6,21]. It has been suggested that glucocorticoid-evoked apoptosis of Jurkat cells requires a repressive function of GR, involving interactions with c-Jun [24]. In CEM-C7 cells, a strong correlation between GR and c-Jun transcription has been reported to facilitate functional cross-talk between the two pathways [25].

With accumulating evidence suggesting a significant role for c-jun in apoptotic pathways, there is increasing interest in understanding how c-jun is regulated. In general, the regulation of c-jun is highly complex and varied in different cell types, as well as with different stimuli. The regulation can be purely transcriptional, post-transcriptional, or both [26–30]. Glucocorticoid induces c-jun expression in some cells, but suppresses it in others [25,31–33]. In both AtT-20 pituitary tumor cells and U-937 monocytic leukemia cells, Dex represses the transcription of c-jun [25,34]. Interestingly, both cell lines resist Dex-dependent apoptosis. In CEM-C7 cells, on the other hand, the results from our previous work and this paper demonstrate that the expression of c-jun is induced by Dex in a time-, dose- and receptor-dependent manner. These cells are killed apoptotically by Dex. The receptor-deficient ICR-27 subclone shows no cell death, growth inhibition, or induction of c-jun. Nuclear run-on assays showed that the induction was transcriptional, and estimate of mRNA half-life showed no effect of Dex treatment.

Jun induction clearly precedes the onset of apoptosis in CEM cells, and we postulate that this sustained induction is relevant to the ultimate cell death [6]. When resting cells are stimulated to grow, a well-documented brief increase in c-jun expression occurs, but prolonged induction of c-jun may be harmful. In CEM-C7 cells, Jun is clearly elevated by 12 h and beyond, after adding Dex to the cells. To determine the role of caspases in their ultimate apoptosis, we have carried out detailed studies of their activity and relationships to the final outcome. This complete set of data is too extensive to present fully in this paper, but we do include (Fig. 3) data germane to our hypothesis, showing the chronology of altered activity of several caspases. We found that caspase 1, known to respond to activation of Fas or TNF-like receptors, was not activated by Dex in these cells. Caspases 2 and 3, ‘effector’ caspases, were activated, however, after the induction of Jun, and just as overt apoptosis begins. This timing is consistent with our hypothesis that the elevation of Jun is important for the eventual recruitment of the caspase cascade, which we have documented elsewhere [35].

The time course of c-jun mRNA expression and the data from nuclear run-on experiments have shown that c-jun induction only began at ≥6 h after addition of Dex to the culture. It is well known that only a few minutes are required for activated GR to enter the nucleus and bind to a simple promoter regulating gene expression [12,19]. This implies that the regulation of c-jun expression by Dex most likely is not by the mechanism of directly and solely binding of GR to the c-jun promoter. Furthermore, there are no classic palin-dromic glucocorticoid response elements (GRE) in the known portions of the c-jun gene’s regulatory region. Although a putative GRE site has been mentioned to exist at the 5% end of the −1.6 kbp region of c-jun promoter [36], no species and sequence information are available, nor have any functional tests of it been reported. In short, Dex induction of c-jun is more likely to be a secondary rather than a primary response.

Since concomitant protein synthesis is typically required for secondary response mechanisms [19], the protein synthesis inhibitor CHX was employed. CHX itself, however, was found to induce c-jun mRNA levels higher than did Dex alone in an equivalent time. This created obvious difficulty in interpreting whether CHX alters the effect of Dex on the expression of c-jun. Washing out CHX after a period of exposure led to the rapid reversal of its inductive effect. Keeping Dex present continually during the time of exposure to CHX and the subsequent period following CHX wash-out resulted in induced c-jun mRNA levels equivalent to those reached by continuous exposure to Dex without CHX. This could occur either because the Dex induction was insensitive to CHX and proceeded unabated through the + CHX period, or because the CHX did indeed block Dex induction, but left the cells in a state such that after CHX removal, induction by Dex was more rapid. We therefore tested the effect of CHX on the Dex induction of c-jun mRNA transcription more directly, by nuclear run-on assays.

In the presence of CHX, Dex could only slightly induce new synthesis of c-jun mRNA, to a level that was significantly less than that caused by Dex alone. This clearly indicated that CHX prevented much of the inductive effect of Dex on c-jun transcription. It seems that de novo protein synthesis was required for induction of c-jun gene by Dex in these cells, a classic ‘secondary response.’ Since ongoing protein synthesis was necessary, relatively labile proteins must be required for Dex-regulation of the c-jun gene. This was not an effect relevant to general protein synthesis machinery, because the rate of transcription of the normalizer housekeeping gene measured was untouched. A search for the relevant primary gene(s) seems warranted.

Our results do not distinguish a pure secondary response from a recently proposed delayed primary response mechanism [19]. A classic secondary response consists of inducer 1 inducing an independent inducer 2 that is solely responsible for the induction of the measured gene product. In a delayed primary response, there is still induction of inducer 2, but both inducers then participate in the induction of the measured mRNA. Since GRs frequently act in conjunction with other transcription factors, a delayed primary response is possible. This possibility is the more likely since our preliminary data (not shown) indicate that the continued presence of Dex was necessary for maintaining the transcription regulation of the c-jun gene. Further supporting such a mechanism is the coordinate regulation of the GR and c-jun, which has been suggested by Vig et al. [25]. Both the mRNA and protein of GR are increased by glucocorticoids in CEM cells. Nuclear run-on experiments have also demonstrated that the gr gene is regulated at the transcriptional level (data not shown). Newly synthesized c-Jun, GR or both proteins might function as activators for transcription regulation of both c-jun and gr genes. All these data suggest that there is a coordinate regulation between the gr and c-jun genes after addition of Dex to CEM cells.

Acknowledgements

This research was conducted in conjunction with the Walls Medical Research Foundation. The authors thank Betty H. Johnson for help in preparing the manuscript.

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