Activity of the GR in G2 and Mitosis

G. ALEXANDER ABEL, GABRIELA M. WOCHNIK, JOE¨ LLE RU¨ EGG, AUDREY ROUYER, FLORIAN HOLSBOER, AND THEO REIN Max Planck Institute of Psychiatry, Munich D-80804, Germany

To elucidate the mechanisms mediating the re- DEX-inducible in G2. Even in mitotic cells, only the ported transient physiological glucocorticoid re- stably transfected MMTV promoter was repressed, sistance in G2/M cell cycle phase, we sought to whereas the same promoter transiently trans- Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 establish a model system of glucocorticoid-resis- fected was inducible. The use of Hoechst 33342 for tant cells in G2. We synchronized various cell lines synchronization in previous studies probably in G2 to measure dexamethasone (DEX)-induced caused a misinterpretation, because we detected transactivation of either two endogenous promot- interference of this drug with GR-dependent tran- ers (rat tyrosine aminotransferase and mouse me- scription independent of the cell cycle. Finally, GR tallothionein I) or the mouse mammary tumor virus activated a simple promoter in G2, excluding a (MMTV) promoter stably or transiently transfected. functional effect of cell cycle-dependent phos- To circumvent the need for synchronization drugs, phorylation of GR, as implied previously. We con- we stably transfected an MMTV-driven green fluo- clude that GR itself is fully functional throughout rescent protein to directly correlate DEX-induced the entire cell cycle, but GR responsiveness is re- transactivation with the cell cycle position for each pressed in mitosis due to chromatin condensation cell of an asynchronous population using flow cy- rather than to specific modification of GR. tometry. Surprisingly, all promoters tested were (Molecular Endocrinology 16: 1352–1366, 2002)

LUCOCORTICOIDS ARE INVOLVED in regula- Conversely, transactivation of GR-responsive promoters Gtion of various biological processes, including is presumed to be cell cycle-dependent since more than development and reproduction, cell growth and pro- 30 yr. One of the first observations was a cell cycle- liferation, and metabolism of carbohydrate, lipids, and dependent induction of tyrosine aminotransferase (TAT) protein (1–3). As a family member of ligand-dependent by glucocorticoids in hepatoma cells (17). While cells transcription factors (4, 5) the GR is activated by hor- were glucocorticoid-responsive during the G1 and S cell mone binding. GR is subsequently translocated into cycle phases, cells in G2 as well as in M were reported to the nucleus, where it binds to specific DNA sequences be completely resistant to glucocorticoids. Moreover, termed glucocorticoid-responsive elements (GREs) induction of endogenous alkaline phosphatase (18) or and increases transcription from nearby promoters. later on of epidermal growth factor receptors (19) in HeLa Several cofactors are important for proper function of cells was shown to be most effective in late G1 and GR. Molecular chaperones in the cytosol keep the S-phase with an apparent lack of glucocorticoid respon- receptor in a conformation capable of binding to hor- siveness during G2/M and early G1. More recently, it was mone with high affinity (6). Chaperones may also be demonstrated that in fibroblasts synchronized in G2 the involved in nuclear translocation of GR (7–9). Several stably transfected mouse mammary tumor-virus (MMTV) nuclear cofactors of GR are also necessary for chro- promoter as well as the endogenous metallothionein-I matin remodeling of nucleosomally organized promot- (MT-I) promoter could not be activated by glucocorti- ers and for efficient interaction with the basal tran- coids (20). However, in accordance with the hypothesis scriptional machinery (10, 11). Important effects of GR of specific glucocorticoid resistance in G2 the MT-I pro- are not mediated by transactivation of target promot- moter was still inducible by heavy metals in G2. Remark- ers, but by transrepression (12, 13). Transrepression is ably, inhibition of GR seemed to be confined to trans- independent of DNA binding (14) but requires interac- activation, because transrepression by GR was not tion with other transcription factors such as activator affected in G2 cells (21). protein 1 or nuclear factor ␬B (15, 16). Despite several observations relating to cell cycle- It is a long-standing observation that glucocorticoids dependent activity of GR, including different hormone exert antiproliferative effects in most cellular contexts. binding during the cell cycle (19, 22, 23) or decreased nuclear translocation of GR in G2 (19, 20), the molec- Abbreviations: CHO, Chinese hamster ovary; CMV, cyto- megaly virus; DEX, dexamethasone; FACS, fluorescence-ac- ular mechanisms leading to G2 silencing of GR func- tivated cell sorter; gal, galactosidase; GFP, green fluorescent tion remained largely unclear. It has been speculated protein; GRE, glucocorticoid-responsive elements; HOE, that differential phosphorylation of GR throughout the Hoechst 33342; Luc, luciferase gene; MTV, mammary tumor cell cycle (24, 25) might contribute to or account for virus; MMTV, mouse mammary tumor virus; MT, metallothionein; p, plasmid; PI, propidium iodide; TAT, tyrosine amino- cell cycle-dependent function of GR. Indeed, rat GR transferase; TK, thymidine kinase. was shown to be a target for cyclin-dependent kinases

1352 Abel et al. • GR Activity in G2 and Mitosis Mol Endocrinol, June 2002, 16(6):1352–1366 1353

and mitogen activated protein kinases in vitro (26). However, using site-specific mutations of phosphorylation sites of GR, it was not possible to identify a distinct phosphorylation pattern of GR that would lead to complete silencing of the receptor (27, 28). Inter- estingly, some functional consequences of GR phosphorylation were found, but these effects turned out to be promoter specific because they were apparent only at simple promoters containing just one to three GREs, but not at complex promoters like the MMTV promoter

(29–31). Complex promoters are able to recruit addi- Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 tional cofactors, which themselves might be cell cycle- dependently regulated (32–34). With the aim to identify molecular mechanisms explaining glucocorticoid resistance in G2, we sought to establish an experimental model to measure cell cycle-dependent glucocorticoid resistance. We tested cell cycle-dependent transactivation of endogenous as well as exogenous glucocorticoid-sensitive pro- Fig. 1. Induction of TAT Activity in G2 and Mitosis moters, stably or transiently transfected in various cell At time zero, asynchronously proliferating H4-II-E-C3 cells lines. To our surprise, we found no silencing of GR were stimulated with DEX (5 nM) in the presence of colcemid function in G2 at all. Furthermore, mitotic repression of (300 nM) and 6-3H-thymidine (0.1 ␮Ci). At the indicated times GR-induced transcription apparently is due to general after stimulation, mitotic cells were harvested by shaking chromatin condensation, and not to specific inactiva- them off and TAT activity and 3H-thymidine incorporation tion of GR. were determined. In parallel, aliquots of asynchronously proliferating cells were stimulated with DEX in the presence of colcemid and prepared at the same times for control. Gray RESULTS bars represent the DEX-induced TAT activity of G2 cells (relative to vehicle-treated cells) as a function of their position in the cell cycle at the time of DEX induction (i.e. the time Induction of TAT by Dexamethasone (DEX) in before mitosis) compared with asynchronously proliferating Hepatoma Cells in G2 or Mitosis control cells (white bars). Data are given as mean Ϯ SEM of three independent experiments each performed in triplicate. The first systematic investigation of cell cycle-depen- The time course of 3H-thymidine incorporation is given up to dent function of GR reported that induction of endog- 11.5 h after time zero (from right to left). enous TAT in cultured rat hepatoma cells by DEX is completely repressed in the G2 phase of the cell cycle and in mitosis (17). Using the same methodical ap- nonsignificant decrease of TAT induction by DEX com- proach, we first checked whether this original finding pared with asynchronously proliferating cells stimu- is reproducible in our cells endogenously expressing lated under the same conditions in the presence of GR and TAT. Asynchronously proliferating rat hepa- colcemid (Fig. 1). toma H4-II-E-C3 cells were incubated with colcemid, The TAT activities of the noninduced reference cells 6-3H-thymidine and DEX at time zero. Every 90 min, also provide no indication for a decrease of the ligand- mitotic cells were harvested for determination of TAT- independent activity of GR in G2. On the contrary, activity as reporter of GR-induced transcription and there is even a small increase from asynchronously 6-3H-thymidine incorporation as a control marker for proliferating cells to G2 cells (1.5- to 1.6-fold for each cell cycle position of cells at the beginning of induction time point; data used for Fig. 1, but not displayed). (time zero). The rationale of the procedure is that cells The slight decrease in inducibility in G2 cells might collected in mitosis a certain time after stimulation either indicate an only partial repression of GR reac- should have started in the cell cycle phase corre- tivity or, more likely, reflect simply the fact that these sponding to the time before mitosis. For example, cells cells were synchronized in mitosis before preparation. harvested in mitosis after 3 h are expected to have To address this issue, H4-II-E-C3 cells were synchro- started in G2 cell cycle phase at the time of induction nized in G2 by incubation with nocodazole after 48 h of (zero). In our experiment, cells harvested after6hand serum deprivation. For synchronization analysis of a later showed significant incorporation of 6-3H-thymi- population grown in parallel under the same condi- dine indicating the expected appearance of cells hav- tions, mitotic cells were shaken off and discarded ing been in S-phase at the time of induction (Fig. 1). In before cell harvesting to eliminate metaphase cells contrast, cells collected after 1.5–4.5 h showed mini- and to obtain cells synchronized in G2. Fluorescence- mal incorporation of 3H-thymidine and, therefore, were activated cell sorter (FACS) analysis confirmed syn- regarded as cells in G2 between time zero and 1.5–4.5 chronization of H4-II-E-C3 cells by nocodazole in G2 h thereafter. These G2 cells showed only a slight, to about 77% (Fig. 2B) compared with about 14% cells 1354 Mol Endocrinol, June 2002, 16(6):1352–1366 Abel et al. • GR Activity in G2 and Mitosis

creased TAT activity of nonstimulated cells in G2 (1.2- fold compared with asynchronous cells, Fig. 2D). In mitotic cells TAT induction by DEX was almost completely abolished (1.2-fold over basal); this is in accordance with the expected repression of DNA transcription during mitosis. These data suggest that there is only little reduction of GR inducibility in H4-II-E-C3 cells in G2, in stark contrast to data in the literature that indicate an almost complete inhibition (17).

Cell Cycle-Dependent Transactivation of the Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 MMTV Promoter in Randomly Proliferating Cells

To evaluate whether the reported G2 silence of GR may be observed with some promoters, but not with others, we decided to use the well-described MMTV promoter. GR inactivity in G2 has been observed at this promoter (20, 21). In addition, we were concerned about potential nonspecific effects by the use of synchronization drugs. Thus, to establish a system that allows to determine simultaneously transcriptional activity and cell cycle phase in individual cells, we stably integrated a mammary tumor virus (MTV)-green fluorescent protein (GFP) construct into HT-22 cells to obtain the cell line HT-22-GFP. HT-22 is a murine neuronal cell line endogenously expressing GR (35). Using FACS analysis, it was possible to measure GFP fluorescence and propidium iodide (PI) fluorescence, i.e. DNA content, simultaneously for each cell of a randomly proliferating cell population, thereby directly correlating transcriptional activity with the position in the cell cycle. Figure 3 shows the MMTV-driven GFP expression correlated with the DNA content of asyn- Fig. 2. TAT Activity of H4-II-E-C3 Cells Synchronized in G2 chronously proliferating HT-22-GFP cells before stim- or in Mitosis ␮ Cells were synchronized in G2 using nocodazole or in ulation (Fig. 3A) and after stimulation with 1 M DEX ␮ mitosis using colcemid. After 8 h, TAT activity was deter- (Fig. 3, B and C). Stimulation with 1 M DEX for 8 h mined for DEX-stimulated and control cells. The histograms resulted in an about 4-fold increase of mean GFP show representative FACS analyses of the cell cycle of asyn- fluorescence (Fig. 3B). By gating the population of chronously proliferating cells (A), nocodazole-treated (B), and interest, we determined the DEX-induced increase of colcemid-treated cells (C) before stimulation. The TAT activity mean GFP fluorescence within the G1, S and G2 gate, (arbitrary scale with noninduced, asynchronous cells set as 1) respectively. With the inducibility of cells within the G1 Ϯ of asynchronous, G2 and M cells is given as mean SEM of gate set as 100%, we calculated the relative increase five independent experiments each performed in duplicate of GFP fluorescence of the S and G2 gate relative to (D). Black bars represent noninduced cells; gray bars, DEX- that in G1. Figure 3D shows that there is no difference stimulated cells. in glucocorticoid responsivity between the three populations after stimulation with DEX for 8 h. The fact that in G2 within the asynchronously proliferating control cells in G2 after the 8-h period of DEX-induction were population (Fig. 2A). Likewise, mitotic cells were col- in the S-phase at the beginning of the induction could lected by synchronization with colcemid and shaking obscure a potential G2 effect, if the reporter gene off. FACS analysis showed more than 95% tetraploid product was stable for at least 8 h. Therefore, we also mitotic cells in these preparations (Fig. 2C). After syn- performed DEX-inductions for only 3 h (Fig. 3C). The chronization, cells were stimulated with DEX (5 nM) for increase of mean GFP intensity was about 2-fold, but 8 h, mitotic cells were discarded and the remaining again stimulation was uniform for G1, S, and G2 (Fig. cells harvested for determination of TAT activity. Asyn- 3, C and D). chronously proliferating cells revealed 5.6-fold TAT We observed a steady increase of GFP reporter with activity compared with nonstimulated control cells increasing PI fluorescence leading to an almost 2-fold (Fig. 2D). Cells synchronized in G2 showed almost the higher mean GFP fluorescence in G2 than G1 cells, same TAT activity after DEX-stimulation as asynchro- both in nonstimulated (Fig. 3A) and stimulated cell nous cells but a slightly diminished inducibility (4.9- populations (Fig. 3, B and C). This increase can be fold over basal). This is mainly due to a slightly in- explained either by a continuous accumulation of re- Abel et al. • GR Activity in G2 and Mitosis Mol Endocrinol, June 2002, 16(6):1352–1366 1355 Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021

Fig. 3. Correlation of DEX-induced MMTV Activity and Cellular DNA Content The MMTV-driven GFP expression of each single HT-22-GFP cell, determined as fluorescence at 525 nm, is given as function of the DNA content, determined as PI-fluorescence at 630 nm. A, Representative cell population before stimulation; B, 3 h; C, 8 h after stimulation with DEX. G1, S, and G2 denote the positions of the gates of the corresponding phase of the cell cycle. D, Values of relative GFP expression induced by DEX within the S and G2 gate were normalized to the value of inducibility of GFP expression within the G1 gate and are given as mean Ϯ SEM of four independent experiments performed in triplicate. porter between each cell division or by an increase in HT-22-GFP cells were subjected to serum starvation GR number in G2, which could give rise to an increase (0.5% FCS) for 48 h followed by at least 18 h release of both, ligand-dependent and -independent activity. in medium containing (10% FCS) in the absence (con- We also noted an increase in the proportion of G1 cells trol) or presence of either nocodazole or taxol. Aliquots after8hofincubation with DEX (Fig. 3B). This is were taken to confirm synchronization using FACS consistent with an extended G1 phase, probably due analysis. Mitotic cells were discarded before as de- to some unknown effect of DEX in this cell cycle scribed above. Cells were treated with 1 ␮M DEX vs. phase. It is unlikely that this would affect our measure- ethanol for 8 h and then processed for measurement ment of G2 cells. G1 cells also appear readily stimu- of MMTV-driven GFP expression by FACS. Treatment lated by DEX. Moreover, we did not observe significant with nocodazole resulted in synchronization in G2 of changes in growth behavior of these cells upon incu- on average 86% of the cell population (Fig. 4B) com- bation with DEX for 1–3 d (not shown), suggesting a pared with 12% cells in G2 in the absence of synchro- transient effect of DEX on the G1 population. Taken nizing agent (Fig. 4A). With taxol, 80% of the cells were together, our data of the MMTV promoter in nonsyn- synchronized in G2 (Fig. 4C). By gating of either the chronized cells provide no support for transcriptional inactivity of GR in G2. entire cell population or cells in G2 (Fig. 4, A–C) we determined the cell cycle-dependent transactivation Transactivation of the MMTV Promoter in Cells of the MMTV promoter. It is obvious from Fig. 4D that Synchronized in G2 also in these G2 cell preparation there is no reduced response to DEX. The small, nonsignificant increase in To address the possibility that a potential silencing of stimulated G2 cells as compared with asynchronous GR in G2 was obscured even at the short DEX-induc- cells is similarly observed in the GFP fluorescence of tion time of3hwesetouttoverify our data with the nonstimulated cells: nocodazole and taxol arrested MMTV promoter by using G2 synchronizing drugs. cells each display a 1.5-fold higher reporter level. This 1356 Mol Endocrinol, June 2002, 16(6):1352–1366 Abel et al. • GR Activity in G2 and Mitosis

(36): condensation of chromatin or modification of distinct transcription factors (37–39). We were interested to find out what mechanism may apply to GR-dependent transcription. First, we issued whether GR-mediated transcription is impaired during mitosis. We decided to use the MT-I promoter in HT-22 cells, because this endogenously active promoter is inducible by glucocorticoids and by heavy metals. This allowed us to check GR-independent inducibility of the promoter. Moreover, this pro-

moter also was used to show GR silence in G2 (20). Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 Therefore, we additionally checked for transactivation of MT-I by DEX in G2. HT-22 cells were synchronized in G2 using nocodazole or in mitosis using colcemid as described above. After synchronization, cells were ␮ ␮ stimulated either with 1 M DEX or 5 M CdCl2 for8h. Transcriptional activity of the MT-I promoter was determined using quantitative RT-PCR. The amount of amplified MT-I mRNA (280 bp) was normalized to the intensity of the corresponding ␤-actin mRNA. Colce- mid treatment resulted in over 95% accumulation of tetraploid cells (Fig. 5A). Transcription from the MT-I promoter in asynchronously proliferating cells increased on average 3.34-fold Ϯ 0.78 (n ϭ 5) over basal activity by DEX (representative gel in Fig. 5B, lane 1 and 2) and on average 5.47-fold Ϯ 1.60 (n ϭ 5) by

CdCl2 (lane 3). Cells synchronized in G2 showed no significantly altered MT-I transcription neither by DEX Ϯ ϭ nor by CdCl2 (DEX 2.43-fold 1.36, n 5, and CdCl2 5.49-fold Ϯ 1.79, n ϭ 5, respectively, Fig. 5B, lanes 4–6). Cells synchronized in mitosis showed a negligi- ble induction of MT-I by DEX, but still a noticeable Fig. 4. MMTV Activity of Cells Synchronized in G2 induction by CdCl (DEX 1.15-fold Ϯ 0.38 and CdCl Asynchronously proliferating (A), nocodazole-synchro- 2 2 3.28-fold Ϯ 1.60, both n ϭ 5 activity over basal value; nized (B), or taxol-synchronized (C) HT-22-GFP cells were analyzed by FACS before stimulation. MMTV activity was Fig. 5B, lanes 7–9). Noninduced cells show no differ- determined as DEX-induced GFP expression relative to non- ence in the amount of MT-I mRNA before induction induced cells by either gating the entire asynchronously pro- (G2 cells 1.07-fold Ϯ 0.19 compared with asynchro- liferating cell population (A) or solely G2 cells (B, C) and is nous, M cells 0.99 Ϯ 0.54; n ϭ 5). These data confirm presented as mean Ϯ SEM of five independent experiments that cells in G2 are not glucocorticoid resistant, performed in triplicate (D). Black bars represent noninduced whereas induction of transcription by glucocorticoids cells; gray bars, DEX-stimulated cells. is repressed in mitotic cells, probably through chromatin condensation. is also reflected in a virtually unchanged stimulation To answer the question whether mitotic repression of after DEX (4.37 Ϯ 0.34-fold for asynchronous cells, a glucocorticoid responsive promoter is mediated solely 3.86 Ϯ 0.21-fold for nocodazole-arrested cells, and by mitotic condensation of chromatin, HT-22-GFP cells 3.54 Ϯ 0.21-fold for taxol-arrested cells; data used for stably expressing MMTV-driven GFP were transiently Fig. 4, but not displayed). cotransfected with the same promoter linked to another In sum, our data obtained from the TAT promoter in reporter, i.e. the luciferase gene (pMTV-Luc). The MMTV hepatoma cells as well as from the MMTV promoter in promoter is organized into a phased array of nucleo- neuronal cells indicate that the cell cycle-dependent si- somes when stably integrated into cellular DNA (40), lencing of GR function reported in previous publications whereas the same promoter transiently transfected re- is not reproducible in our cell systems and, therefore, at mains in an unorganized state (41–43) and is excluded least, may not be a common principle of regulation of GR from mitotic condensation of nuclear chromatin. There- function. We next asked whether silencing of GR can be fore, transcription from the transient template in mitosis found in the phase after G2, mitosis. should only be affected if transcription factors like GR are inhibited directly. GR Activity in Mitosis on Transient Templates Cells were synchronized in G2 or M. During the synchronization procedure, HT-22-GFP cells were Two explanations are provided, in general, for the transiently cotransfected with pMTV-Luc and pCMV nonspecific repression of DNA transcription in mitosis (cytomegalovirus)-␤-galactosidase (gal). While in G2 Abel et al. • GR Activity in G2 and Mitosis Mol Endocrinol, June 2002, 16(6):1352–1366 1357

phosphorylated (24). In addition, it has been con- cluded that only simple glucocorticoid-responsive promoters are functionally sensitive to GR phosphorylation, but not complex promoters like the MMTV (29–31). Therefore, we asked whether GR silence in G2 might exist for simple promoters. HT-22-GFP cells or H4-II-E-C3 cells were transiently transfected with a simple promoter construct, a truncated thymidine kinase (TK) promoter linked to a single GRE and to the luciferase gene (pTK-GRE-Luc). Cells were synchro-

nized in G2 as described and stimulated with DEX for Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 8 h. In both cell lines, the promoter was equally inducible in G2 and in asynchronous cells (Fig. 7). Nonin- duced G2 cells again showed a small, nonsignificant increase in reporter activity (1.3-fold for HT22 cells and 2-fold for H4-II-E-C3 cells, data used for Fig. 7, but not shown). This confirms that phosphorylation of GR has at least no direct effect on transactivation of glucocorticoid sensitive promoters. Moreover, GR is active in G2 on both complex and simple promoters.

GR-Induced Transactivation in Cells Treated with Hoechst 33342 (HOE)

Looking for methodological differences between our experimental set-up and previous reports showing G2 silence of GR we noted that previous studies had used HOE to synchronize cells in G2 (20, 21). We initially refrained from using this drug, because it is known as topoisomerase inhibitor (44), nonspecific inhibitor of transcription (45, 46), and a toxic agent for some cell types (47, 48). Nevertheless, it was necessary to test whether the described GR silence in G2 depends on the way cells are synchronized in G2. Fig. 5. Inducibility of the MT-I Promoter by DEX or CdCl2 in Therefore, we began by testing the efficiency of Either G2 or Mitosis HOE to synchronize our cells in G2. It first should be HT-22-GFP cells were synchronized in G2 by nocodazole noted that HT-22-GFP cells we had used for this study or in mitosis by colcemid (A). Asynchronous and synchro- were not efficiently synchronized in G2 by treatment nized cells were stimulated for 8 h with either DEX or CdCl . 2 with HOE (data not shown). The only cell line that in our MT-I mRNA was determined by RT-PCR. B, Representative agarose gel showing the RT-PCR products of MT-I and ␤-ac- hands readily synchronized in G2 after HOE treatment tin mRNA of asynchronous cells or cells synchronized in G2 was Chinese hamster ovary (CHO)-TRex (Fig. 8A). or mitosis. Ctrl, Noninduced; DX, DEX-induced; Cd, CdCl2- These cells are a commercially available derivative of induced, ␾, ␾X-HaeIII DNA molecular weight marker. CHO cells, which have been reported to synchronize in G2 by treatment with HOE (49). After pretreatment with hydroxyurea and 18 h incubation with HOE, DEX-in- both MMTV templates were inducible, in mitosis the duced transactivation of the transiently transfected transiently transfected MMTV promoter was inducible MMTV promoter was indeed not detectable in these and the stably integrated one was silent (Fig. 6). The cells (Fig. 8B). However, DEX response was not af- reporter activities of noninduced cells showed no sig- fected in G2 CHO-TRex cells synchronized by no- nificant difference (compare black bars). These data codazole (Fig. 8, A and B). To explore the differences show that mitotic repression of GR-dependent tran- between the synchronization procedures, we treated scription is likely due to chromatin condensation rather cells with different concentrations of HOE for 12 h than to modification (e.g. by phosphorylation) of GR or without the preceding hydroxyurea incubation. Again, any cofactor of GR. DEX response was not measurable at a concentration of 6 ␮g/ml HOE (Fig. 9A). However, FACS analysis Transactivation of a Simple Glucocorticoid- revealed that these cells were not synchronized in G2 Responsive Promoter in G2 Cells (Fig. 9B). This strongly suggested that the inhibition observed with HOE treatment is not due to synchro- GR was reported not only to be differentially active nization in G2, but to some cell cycle-independent throughout the cell cycle, but also to be differentially effect of HOE. Paradoxically, while at low concentra- 1358 Mol Endocrinol, June 2002, 16(6):1352–1366 Abel et al. • GR Activity in G2 and Mitosis Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021

Fig. 6. Comparison of Transiently with Stably Transfected MMTV Promoter Templates in G2 or in Mitosis HT-22-GFP cells were transiently cotransfected with pMTV-Luc and pCMV-␤-gal and treated with DEX or ethanol only, either without synchronization or after synchronization in G2 or mitosis. After harvesting, aliquots of each sample were either processed for determination of GFP fluorescence using FACS analysis (right panel) or for determination of luciferase and ␤-gal activities (left panel). GFP expression and luciferase activity (normalized to ␤-gal activity, which was similar for asynchronous, G2, and M phase cell populations) after DEX-treatment (gray bars) are given in comparison with ethanol-treated samples (black bars) as mean Ϯ SEMR of five independent experiments each performed in duplicate. ***, P Յ 0.005.

To corroborate these findings, HT-22-GFP cells were coincubated with DEX and increasing concentrations of HOE ranging from 0.25 up to 4 ␮g/ml for 8 h. Although the cell cycle of these cells was not affected by HOE (Fig. 10A), induction of the MMTV promoter by DEX was dose dependently inhibited (Fig. 10B). In contrast to the transiently transfected MMTV promoter in CHO-Trex cells, the transcription from the stably integrated MMTV promoter in the absence of DEX was not significantly increased in HT-22-GFP cells (Fig. 10C). However, when we transiently transfected these cells with the MTV-Luc construct, again the promoter activity was significantly enhanced by HOE in the absence of DEX (Fig. 10D). Fig. 7. DEX-induced Transactivation of a Simple Promoter in Thus, although we did not further analyze the mech- Cells Synchronized in G2 anism leading to inhibition of GR-mediated transacti- HT-22-GFP or H4-II-E-C3 cells transiently transfected with vation by HOE in this study, we note a paradoxical pTK-GRE-luc were synchronized in G2 using nocodazole and ␮ difference in the effect of low concentrations of HOE stimulated with DEX (1 M) for 8 h. Luciferase activity was ␮ normalized to the ␤-gal activity of the cotransfected pCMV- (about 1–4 g/ml) on transiently vs. stably transfected ␤-gal. The DEX-induced luciferase activity is given for each MMTV promoter templates in the absence of hor- cell line relative to ethanol-treated cells as mean Ϯ SEM to- mone. While transcription from the stably integrated gether with the numbers of independently performed exper- template is not affected (Fig. 10C), transcription from iments. transient templates is enhanced (Figs. 9A and 10D). With concentrations of HOE as high as in previous reports [7.5 ␮g/ml (20)] MMTV promoter activity was tions of HOE there is still inducibility of the MMTV always down, with or without DEX and independent of promoter, we observed a significant increase of the synchronization (not shown). nonstimulated MMTV activity at 1 and 2 ␮g/ml of HOE (Fig. 9A). We wondered whether the stimulating effect of HOE is observed at GR-independent promoters. We mea- DISCUSSION sured the activity of transiently transfected ␤-gal driven by a tetracyclin-inducible CMV promoter in The initial aim of our study was to identify new mech- CHO-TRex in the presence or absence of HOE. Tran- anisms that could explain glucocorticoid resistance as scriptional activity was not significantly elevated it occurs under certain physiological and pathological with increasing concentrations of HOE (Fig. 9C), sug- conditions. Because we aimed at processes that may gesting a somewhat specific effect of HOE on GR- pertain to GR function in vivo, we decided to make use dependent promoters. of the transient GR silence within the physiological Abel et al. • GR Activity in G2 and Mitosis Mol Endocrinol, June 2002, 16(6):1352–1366 1359 Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021

Fig. 8. MMTV Promoter Activity of CHO-Trex Cells Synchronized by Nocodazole or HOE Asynchronously proliferating cells were presynchronized with hydroxyurea, transiently transfected with MTV-Luc reporter plasmid and released into medium containing either nocodazole or HOE (6 ␮g/ml). The histograms in A show representative results of cell synchronization compared with asynchronously prolifareting cells. B, Corresponding transcriptional activation. Synchronized cells were stimulated with DEX and tetracyclin for 12 h. Data are presented as mean values Ϯ SEM of MTV-Luc activity normalized to the ␤-gal activity of the cotransfected pCMV-␤-gal from four experiments independently performed in duplicate. Black bars, Ethanol control; gray bars, DEX induction. context of the cell cycle, which has been reported of TAT inducibility in G2 cannot be explained by non- previously (17–21). specific effects of cytoskeleton disrupting agents like However, from our extensive studies of four different colcemid or nocodazol, because we found that they promoters in three different cell types, we conclude did not affect GR function neither in asynchronously that GR is active throughout the entire cell cycle, proliferating H4-II-E-C3 cells nor in HT-22 cells (data thereby completely changing our view of GR. not shown). This is in accordance with previous re- Inactivity of GR in G2 cells was first reported in 1969 ports regarding nuclear translocation of GR (8, 50). We using the TAT promoter in rat hepatoma cells (17). In cannot rule out the possibility that this slight decrease an effort to reproduce this previous report directly, we reflects a partial impairment of GR in G2 in H4-II-E-C3 investigated the activity of the TAT promoter in H4-II- cells. However, we consider it more likely that this E-C3 rat hepatoma cells. While there was clear repres- minor reduction is explained by the higher proportion sion of TAT induction in mitosis (Fig. 2D), in G2 cells of cells in mitosis in this population as compared with we found no difference in the TAT activity after induc- randomly proliferating cells. tion with dexamethasone and only a marginal reduc- We can only speculate why the previous report (17) tion of TAT inducibility compared with asynchronously is in contrast to our observations: for example, the TAT proliferating cells (Figs. 1 and 2). This slight decrease promoter is regulated not only by glucocorticoids via a 1360 Mol Endocrinol, June 2002, 16(6):1352–1366 Abel et al. • GR Activity in G2 and Mitosis Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021

Fig. 9. Cell Cycle-independent Effect of HOE on GR-Dependent Transcription in CHO-TRex Cells A, Transactivation of MTV-Luc transiently transfected in CHO-TRex cells induced by 1 ␮M DEX in the presence of increasing concentrations of HOE (exposure time 12 h). Bars represent mean values Ϯ SEM of either basal MTV-Luc activity (without DEX) in the presence of HOE (black bars) or stimulated MTV-Luc activity (DEX ϩ HOE, gray bars). Significance is given for black bars (i.e. significant difference of cells in the absence of DEX treated with HOE vs. untreated cells). Luciferase activity was normalized to protein. B, DNA histogram of asynchronously proliferating cells incubated with HOE (6 ␮g/ml) for 12 h. C, Tetracycline-induced ␤-gal activity in CHO-TRex cells in the presence of HOE. Cells were stimulated with 1 ␮M tetracycline in the presence of HOE. *, P Յ 0.05; ***, P Յ 0.005.

GRE (51, 52), but also by other factors like cAMP (53, GR-dependent promoters and because it has been 54), or liver-specific hepatocyte nuclear factors (55, used to show GR silence in G2 before (20, 21). We 56). Activation of TAT transcription by the cAMP path- created HT-22 cells stably transfected with an MMTV- way has been reported to be differentially sensitive in driven GFP gene to be able to directly correlate GR- different liver-derived cell lines downstream of protein dependent transactivation with the cell cycle for each kinase A (57). Therefore, it is possible that cell type- cell in either synchronized cells or unsynchronized, specific and cell cycle-dependent variations in the randomly proliferating cells. A similar methodical ap- cross-talk between these different signal transduction proach was successfully used to show that heat shock pathways may finally lead to impaired transactivation protein 70 is cell cycle-dependently induced after heat of the TAT promoter in G2. This would also mean that stress in a limited number of cell lines (58). In our GR function itself is not directly linked to the cell cycle. HT-22-GFP cells, FACS analysis revealed no cell cycle An alternative, though less likely, explanation would be dependence of DEX-inducibility of GFP expression that it can be difficult to avoid contaminations of G2 (Fig. 3). We noted, however, that cells with or without cell preparations with mitotic cells in the way G2 cells induction with DEX displayed an increasing amount of have been prepared (17). reporter product as they moved from G1 to S and to To either corroborate or disprove our conclusions, G2. We cannot completely rule out that this reflects a we used the MMTV promoter as another model sys- more efficient hormone binding and transactivational tem because it represents one of the best-studied activity of GR during S-phase (17–19, 23), which may Abel et al. • GR Activity in G2 and Mitosis Mol Endocrinol, June 2002, 16(6):1352–1366 1361 Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021

Fig. 10. Differential Effect of HOE on Transiently or Stably Transfected MMTV Promoter Templates A, Representative histogram showing the effect of a short-time (8 h) exposure of asynchronously proliferating HT-22-GFP cells to 1 ␮g/ml HOE. B, Transactivation of HT-22-GFP in the presence of increasing concentrations of HOE. Bars represent mean values Ϯ SEM of MTV-GFP induction by 1 ␮M DEX for 8 h (relative to uninduced cells). C, Basal values of MTV-GFP activity in the presence of HOE. D, Effect of increasing concentrations of HOE on the transactivation of MTV-Luc transiently transfected in HT-22-GFP cells. Bars represent luciferase activity stimulated by DEX at 1 ␮M for 12 h in the presence of HOE (12 h, i.e. added simultaneously with DEX) (gray bars) or luciferase activity in the presence of HOE and the absence of DEX. Significance is given for black bars.*,P Յ 0.05.

seemingly extend into G2, if GFP protein is stable. nize HT-22-GFP cells in G2 and found no GR silencing However, because the increase is independent of in- at all. duction with DEX, the more likely explanation is that it Similarly, promoter specificity is unlikely to be the is due to a steady accumulation of GFP between each cue for explaining the difference between our data and cell division. Similarly, it is possible that the substan- those of others, because we also tested the endog- tially greater mean variation of GFP expression of cells enously expressed MT-I promoter and a simple pro- in G1 reflects differential DEX-responsiveness be- moter construct, again with no evidence for GR silenc- tween early G1 cells and late G1 cells as suggested ing in G2. The MT-I promoter was also used to show elsewhere (25). However, we consider it more likely impaired GR function in G2 (20). The simple promoter that the higher variation in G1 is due to the higher containing only one GRE was of particular interest, proportion of G1 cells in the total population, i.e. cells because on the one hand GR is differentially phos- spend more time in G1 which means that cells late in phorylated during G1/S and during G2/M (24, 25) and G1 had more time to accumulate GFP. The difference on the other hand mutations of certain phosphoryla- of our results to the data describing silencing of GR in tion sites of GR affects transactivation only with simple G2 (20, 21) cannot be explained by the fact that GR glucocorticoid responsive promoters (25, 29–31). silence previously was found in synchronized cells, Thus, one would have predicted that a cell cycle- because we also used nocodazole or taxol to synchro- dependent activity of GR would be detectable only 1362 Mol Endocrinol, June 2002, 16(6):1352–1366 Abel et al. • GR Activity in G2 and Mitosis

with a simple promoter construct, if differential phos- significant fraction of M-phase cells. In addition, it cannot phorylation was causal for it. Because we found no cell be excluded that EGF binding is down-regulated by cycle dependence of GR function also on our simple ways other than reduced transcription. The work by Grif- promoter, we postulate that the cell cycle-dependent fin and Ber (18) has been cited as support for inactivity of phosphorylation of GR has no effect on its ability to GR in G2 in numerous publications (e.g. Refs. 25, 67–70). transactivate. However, although the authors analyzed induction of The discrepancy of our results to those obtained alkaline phosphatase by hydrocortisone for 48 h after previously is, most likely, explained by the use of HOE mitotic cell selection, they did not analyze cells in the G2 in previous studies to synchronize cells in G2 (20, 21). phase. Inactivity in G2 may have been inferred from the We demonstrate that HOE itself interferes with GR- 20 h lag period of induction when cells are exposed to

dependent transcription. The bisbenzimide HOE was DEX 12 h after mitosis (see Fig. 2 in Ref. 18). However, Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 reported to be suitable for reversible cell cycle syn- this is a rather indirect conclusion and the authors them- chronization of CHO cells (49, 59) and, more recently, selves make no claim about G2, they actually never of primary cultured porcine fibroblasts (60). CHO cells mention G2 (18). (49) and L-fibroblasts (20) apparently synchronized In most experiments, we observed a small, albeit well in G2 at a concentration of HOE of 7.5 ␮g/ml. nonsignificant increase in the amount of GR-depen- While our CHO-TRex cells readily synchronized in G2 dent reporter in G2. When observed for each cell (Fig. at a concentration of HOE of 6 ␮g/ml, HT-22-GFP cells 3), this may be explained by a steady increase in failed to synchronize efficiently by HOE treatment, reporter between each cell division (see above). When even after presynchronization in S-phase by hy- normalized by total protein or ␤-gal activity, this in- droxyurea (data not shown). Importantly, we found crease may reflect the reported increase of GR binding that HOE interfered with GR-mediated transactivation sites in S and G2 as compared with G1 (19), an ex- of MMTV in HT-22-GFP cells and CHO-TRex cells planation that would also apply to measurements per even in the absence of cell synchronization (Figs. 9 cell, of course. From Western blotting of protein ex- and 10). Strikingly, while HOE inhibited transcription tracts of asynchronous or G2/M-synchronized HT-22- from a stably integrated MMTV promoter, the same GFP cells, we actually have preliminary evidence for a promoter transiently transfected was activated even in small increase in GR protein in G2 and M-phase (data the absence of DEX (Fig. 10). Although we did not not shown). However, given the about equally small pursue this phenomenon further, we conclude that the increase in ligand-independent reporter activity in G2 use of HOE is not recommendable for assaying GR and unchanged inducibility, we consider it unlikely that function. the increase in GR number in G2 compensates for a It is well described that prolonged exposure to HOE is possible reduction in GR activity per se. toxic to several cell types at nanomolar (48, 61) to While we found no inhibition of GR-dependent tran- micromolar concentrations (62). Using the MTT assay, scription in G2, transcription of the endogenous MT-I we found no cytotoxicity after 8 or 12 h incubation of promoter and the stably integrated MMTV promoter was HT-22-GFP and CHO-TRex cells with HOE (not shown), not inducible by DEX in mitotic cells. With the endoge- but determined a half-maximal cytotoxic concentration nous MT-I promoter, we observed in mitosis only a re- ␮ of HOE for HT-22-GFP cells of about 3 g/ml after 32 h duced, but still evident inducibility by CdCl2. While we do of exposure (data not shown). While we observed inhi- not know the reason for this partial activity even during bition of DEX-response already at nontoxic concentra- mitosis, we note that during mitosis DNase I hypersen- tions, one might speculate that cells are nevertheless sitive sites can persist (38) and even TFIID-promoter already determined for an apoptotic process. This pro- complexes remain stable (71). Therefore, it may be pos-

cess could involve p53, which functionally inhibits GR sible that CdCl2, in contrast to DEX, activates factors that function (63). Another potential mechanism explaining are able to activate the MT-I promoter preassembled inhibition of DEX-induced transcription involves interfer- with TFIID complexes even in mitosis. ence of HOE with DNA binding of transcription factors, In contrast to the stably integrated, chromosomal which has been shown at several examples (45–47, 64). template, the MMTV promoter transiently transfected It may be for these reasons that HOE seems not to be clearly was inducible by DEX even in mitosis. This used as the standard agent for cell synchronization in G2 model system of stable and transient cotransfection of (65, 66). the MMTV promoter at the same time has been suc- While the use of HOE can serve as a possible expla- cessfully employed to elucidate the critical relevance nation for the difference between our conclusions and of nucleosomal organization for GR-mediated tran- those using HOE, we can only speculate about other scription (42, 72–75). We exploited this model system studies (17–19). We discussed Ref. 17 above. Citation here for the first time to shed light on the mechanism (19) reports on the induction of EGF binding by DEX of mitotic inhibition of GR-dependent transcription. treatment throughout the cell cycle. Unfortunately, sam- The hypotheses put forward to explain transcriptional ples have been taken at the S/G2 transition and at the repression during mitosis fall into two general catego- G2/M transition, but none clearly in G2 (Fig. 2 of Ref. 19). ries: hypotheses using chromosome condensation Because G2 and M cells were not differentiated, one and hypotheses using inactivation of transcription fac- might speculate that the cell preparation contained a tors as explanation (36–39). Our findings clearly show Abel et al. • GR Activity in G2 and Mitosis Mol Endocrinol, June 2002, 16(6):1352–1366 1363

that GR itself is not inhibited in mitotic cells, and Cell Synchronization and Induction therefore, repression of GR-dependent transcription in mitosis must be due to the nonspecific effect of chro- Exponentially growing HT-22 and H4-II-E-C3 cells were maintained in DMEM supplemented with 0.5% charcoal- matin condensation. Further, our results exclude the stripped FCS for 48 h. After serum starvation cells were hypothesis that there is bulk inactivation of the basal released into DMEM supplemented with 10% FCS containing transcriptional machinery during mitosis. either 500 ng/ml nocodazole or 100 nM taxol for synchroni- In summary, we propose a model of cell cycle- zation in G2 or containing 300 nM colcemid for synchro- dependent activity of GR, in which GR-dependent nization in mitosis. Cells were maintained in the presence of each synchronizing agent for 18–24 h. After this time, aliquots transcription is silenced only during mitosis. This si- of cells grown under identical conditions were either pre- lencing, however, is due to chromatin condensation pared for FACS analysis to verify synchrony or were stimu-

rather than to inactivation of GR itself. Therefore, chro- lated with DEX or CdCl2. To obtain populations of cells in G2, Downloaded from https://academic.oup.com/mend/article/16/6/1352/2741824 by guest on 29 September 2021 matin-independent functions of GR are not sup- mitotic cells were eliminated from adherent cell cultures treated with either nocodazole or taxol by shaking them off pressed in mitosis, e.g. transcription from viral tem- before FACS analysis or determination of luciferase or TAT plates or, possibly, DNA-independent activities of GR. activity. Conversely, for preparation of mitotic cells, meta- Our model leaves open the possibility that certain phase cells were shaken off from adherent cell populations GR-responsive promoters are regulated in a cell cycle- treated with colcemid before stimulation. dependent manner due to cell cycle-dependent activ- Routinely, all cell lines were cultured in DMEM containing charcoal-stripped steroid-free FCS for at least 24 h before ity of promoter-specific cofactors. hormone treatment. All treatments were done in steroid-free DMEM with either DEX dissolved at the concentrations indicated in ethanol or an identical volume of ethanol. Cadmium MATERIALS AND METHODS chloride was dissolved in water and used at a concentration of 5 ␮M. HT-22-GFP and H4-II-E-C3 cells were routinely exposed to DEX or CdCl for 8 h. CHO-TRex cells transiently Cell Lines and Culture 2 transfected with either MTV-Luc or pTK-GRE-luc were routinely exposed to DEX for 12 h. Mouse neuronal HT-22 cells (35) and CHO-TRex cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, FACS Analysis MD) supplemented with 10% FCS (Biochrom, Berlin, Germa- ny), 50 U/ml each of penicillin and streptomycin, and 4.5 g/liter glucose. CHO-TRex were from Invitrogen (Carlsbad, Cells were harvested by trypsinization and fixed in 70% eth- CA). H4-II-E-C3 rat hepatoma cells (ATCC CRL-1600, Man- anol at 4 C over night. Samples of fixed cells were resus- ␮ assas, VA) were maintained in DMEM devoid of phenol red pended and stained in PBS containing 20 g/ml PI and 10 ␮ supplemented with FCS and antibiotics as above, 1 g/liter g/ml RNAse A. FACS analysis of GFP emission at 525 nm glucose and 2 mML-glutamine. (Fl1) and PI emission at 630 nm (Fl4) was performed using a Beckman Coulter (Krefeld, Germany) XL flow cytometer. After Plasmids and Transfection gating out doublets and clumps as described elsewhere (82), results of MMTV-driven GFP induction in HT-22-GFP cells were obtained from the mean GFP fluorescence of all events The plasmid pRK5MLuc was obtained by cloning the PvuII- within a particular gate, e.g. a G2 gate or a gate spanning the BamHI fragment from pMTV-Luc (76) encompassing the entire cell cycle. Cell cycle analysis of DNA histograms was MMTV-LTR, the firefly luciferase structural gene and the done using the Multicycle software (Phoenix Flow Systems, SV40 polyA signal into the SpeI-AccI vector fragment from San Diego, CA). pRK5SV40PUR (77) containing the puromycin resistance gene. The Acc65I site was blunted and the SpeI site was ligated to the BamHI site via a linker. The plasmid pMTV-GFP Assays of Luciferase, ␤-gal, and TAT Activity was cloned by replacing the luciferase structural gene of pRK5Mluc (excised with XhoI and Acc65I) with the GFP gene Luciferase activity was determined according to manufacturer’s linked to a sorting signal for localization to the endoplasmatic instructions (Roche Molecular Biochemicals, Mannheim, Ger- reticulum, which was amplified by PCR from the plasmid many) using a luminometer [Wallac, Inc. (Wildbad, Germany), ER-GFP kindly provided by D. Pestov (78). victor2 multilabel counter]. Assays of ␤-gal activity were per- The plasmid pTK-GRE containing a single GRE from a formed using the Galacto-Light assay (Tropix, Inc., Bedford, truncated TK promoter linked to the luciferase gene was MA) according to the manufacturer’s instructions. Luciferase kindly provided by D. Spengler (79). Cells were transiently activity obtained from pMTV-Luc transiently transfected in HT- transfected using ExGene 500 (MBI Fermentas, St. Leon-Rot, 22-GFP or H4-II-E-C3 cells was normalized to gal activity of Germany) according to the manufacturer’s instructions. For cotransfected pCMV-␤-gal to correct for variations in transfec- transfection of cells synchronized in G2 or M, transfection tion efficiency. Luciferase activity obtained from pMTV-Luc tran- was at the end of serum starvation before incubation with siently transfected in CHO-TRex cells with or without stimula- either nocodazole, taxol or colcemid (all from Calbiochem, La tion by DEX was normalized to protein. In these cells, inducibility Jolla, CA). of general transcription was determined by induction of pCMV- Electroporation was used to obtain HT-22 cells stably ␤-gal activity by tetracyclin (1 ␮g/ml for 12 h) after normalization transfected with pMTV-GFP (80). Individual cell clones were to protein. grown in the presence of puromycin (10 ␮g/ml) and picked as TAT activity was determined as described (83). In short, 10 described elsewhere (81). One of these subclones was fur- ␮l samples of Triton-X 100-lyzed H4-II-E-C3 cells were incu- ther purified by the criterion of maximally DEX-induced GFP bated in 100 ␮l of a potassium phosphate buffer containing ␮ expression using a fluorescence-activated cell sorter (FAC- 125 mM K2HPO4/KH2PO4,pH7.6;4mML-tyrosine; 70 M Scalibur, Becton Dickinson and Co., Heidelberg, Germany) to pyridoxal-5-phosphate; 13 mM ␣-ketoglutaric acid; and 0.5% obtain the clone HT-22-GFP. CHO-TRex cells stably ex- Triton-X-100 at 37 C for 40 min. After addition of 50 ␮l1% pressing a tetracyclin sensitive repressor protein were tran- hexatrimethyl-ammonium bromide dissolved in 2.8 M NaOH, siently cotransfected with a construct of a tetracyclin induc- samples were incubated at 37 C. After 30 min TAT activity of ible CMV promoter linked to the ␤-gal gene and pMTV-Luc. each sample was assayed by measuring the absorbance at 1364 Mol Endocrinol, June 2002, 16(6):1352–1366 Abel et al. • GR Activity in G2 and Mitosis

340 nm using a plate reader (Dynatech Corp. MR 7000). TAT Received October 12, 2001. Accepted February 8, 2002. activity was normalized to protein. Address all correspondence and requests for reprints to: The relative activity of reporter enzymes was the ratio of Theo Rein, Max Planck Institute for Psychiatry, Kraepelin- stimulated activity divided by nonstimulated activity. The lu- strasse 10, Munich D-80804, Germany. E-mail: theorein@ ciferase signal from pMTV-Luc transiently transfected in mpipsykl.mpg.de. CHO-TRex cells was either normalized to protein or to tetracyclin-induced gal activity normalized to protein. All statistics were performed using the U test to determine significance. REFERENCES

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