Regulation of c-jun and jun-B by Progestins in T-47D Human Breast Cancer Cells

Moussa

Alkhalaf*

and

Leigh

C. Murphyt

Department of Biochemistry and Molecular University of Manitoba Winnipeg, Manitoba, Canada R3E OW3

Biology

To investigate further the molecular mechanisms of progestin regulation of human breast cancer cell growth, we studied the effect of progestins on expression of the protooncogene c-jun and other members of the jun family, jun-B and jump in T-47D human breast cancer cells. The progestin medroxyprogesterone acetate (MPA) increased c-jun mRNA levels in a time- and dose-dependent fashion. Maximal effects were seen after 3 h of treatment with lo-100 nM MPA. Under these conditions, the c-jun mRNA was increased 5.4-fold above the control level. Although the c-jun mRNA level was increased by cycloheximide alone, a further 2.4-fold increase was seen when the cells were treated with MPA in the presence of cycloheximide. The p39 c-jun protein was also increased 3.8fold by this treatment. Maximum levels of p39 c-jun protein were achieved 9 h after treatment, and this level was maintained for at least 24 h. Dexamethasone and dihydrotestosterone did not increase the p39 c-jun protein level under these conditions. However, MPA treatment of T-47D cells resulted in a 55% decrease in overall AP-1 activity, as measured by transient transfection of an AP-l-regulated chloramphenicol acetyltransferase reporter gene. These effects were all reversible by cotreatment with a lofold higher concentration of the antiprogestin RU 488. MPA decreased jun-B mRNA levels 50% 1 h after treatment in T-47D cells. Thereafter, jun-B mRNA levels increased and became elevated (2.5- to 3.5fold) 12, 24, and 48 h after treatment compared to the untreated control values. MPA had little, if any, effect on jun-D mRNA levels in T-47D cells. The data support the conclusion that progestins specifically alter the expression and activity of components that can form AP-1 transcription complexes in T-47D cells. These effects may play a role in progestin-induced growth modulation in human breast cancer cells. (Molecular Endocrinology 6: 1625-1633, 1992) 0888-8809/92/l 625-l 633$03.00/O Molecular Endocrinology Copyright 0 1992 by The Endocrine

INTRODUCTION Progestins appear to have both growth stimulatory (1) and growth inhibitory (2) effects on human breast cancer cells in culture. However, the mechanisms by which these effects are mediated are poorly understood. When progesterone receptor-positive human breast cancer cells are growing optimally in the presence of serum and estrogen, the addition of progestins results in growth inhibition. However, in some cases when these cells are grown under estrogen-free conditions (I), the addition of progestins results in enhancement of growth. Both progestin-induced growth stimulation and inhibition are accompanied by specific cell cycle changes (3). Progestins, therefore, may regulate the expression of genes involved in regulating the cell cycle. Our laboratory has been involved in understanding the mechanisms by which progestins cause growth inhibition. We have investigated previously the effects of progestins on gene expression in T-47D cells under conditions of progestin-induced growth inhibition. The expression of epidermal growth factor, transforming growth factor-a (TGFa), and epidermal growth factor receptor was increased by progestin treatment (4-6) while TGFPl expression was decreased (5). These results suggested that the growth inhibitory effect of progestins cannot be simply explained by the effects of progestins on putative autocrinelparacrine growth factor gene expression in T-47D cells. The response of sensitive breast cancer cells to progestins appears to be complex and may involve an initial growth stimulation, followed by the more dominant effect of growth inhibition. Our observations on the effects of progestin on the expression of c-myc (7), a gene generally associated with cell growth, especially movement in and out of the cell cycle (8) support this notion. Progestin treatment resulted in an initial increase in the c-myc mRNA level, which was maximal at 30-60 min; this was followed by a time- and dose-dependent decrease, and then partial recovery of c-myc mRNA levels. The protein products of the jun family of genes can interact with members of the fos gene family to generate AP-1 complexes (9), which have profound effects on gene regulation. Altered expression of c-jun and c-fos have been found previously to be associated with al-

Society

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tered cell growth due to a variety of growth factors (9). Furthermore, it has been shown recently that some members of the steroid hormone receptor family can interact with AP-1 complexes to modify target gene expression (1 O-l 2). It has also been demonstrated that alteration of fos/jun ratios could result in altered hormone responsiveness of gene expression (13). To further investigate progestin regulation of human breast cancer cell growth, we studied the effects of progestins on expression of the protooncogene c-jun and other members of the jun family, jun-B and jun-D, in T-47D cells.

3-6 h of treatment, although c-jun mRNA was still slightly increased after 12 and 24 h of treatment. As a control for RNA loading, the blots were reprobed with a cDNA for the PRA/calcyclin gene (14), whose mRNA level was unaffected by MPA treatment. The middle panel of Fig. 1 indicates that uneven RNA loading does not explain the MPA-induced increase in c-jun mRNA. Three hours of MPA treatment increased the level of cjun mRNA 5.4 + 1.6-fold (mean + SEM; n = 5). The MPA effect on the c-jun mRNA level was dose dependent, with a detectable increase at 1 nM and a maximal effect occurring at 10 nM (Fig. 2). Effects

RESULTS Effect of Medroxyprogesterone Treatment on c-jun mRNA

Acetate

(MPA)

Treatment of T-47D cells with 100 nM MPA increased c-&n mRNA (Fig. 1). A maximal effect was seen after

I

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11

of Other Compounds

on c-jun mRNA Levels

Since MPA is known to interact with the glucocorticoid receptor in addition to the progesterone receptor, we investigated the effects of other compounds on c-jun mRNA abundance in T-47D cells. Cells were treated with the glucocorticoid dexamethasone, retinoic acid, estrogen, the antiprogestin RU 486, and the progestins MPA, progesterone, and R5020 (Fig. 3A). Only the progestins MPA, progesterone, and R5020 increased the level of c-jun mRNA under these growth conditions. Moreover, the antiprogestin RU 486 had no effect by itself (Fig. 3A, lane 5) but was able to inhibit the effect of MPA on c-iun mRNA in a dose-dependent fashion (Fig. 3B). The antiestrogens monohydroxytamoxifen (Fig. 3A, lane 9) and ICI 164 384 (Fig. 38, lane 5) had no effect on c-jun mRNA under these conditions. Effect of Cycloheximide mRNA Levels

on MPA-Induced

c-jufl

The effect of MPA on c-jun expression in T-47D cells was independent of continuing protein synthesis. The effect of cycloheximide treatment on the ability of MPA to increase c-jun mRNA is shown in Fig. 4. Concentra-

0

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48

(hrs)

1. Time Course of the Effect of MPA on c-jun mRNA Levels in T-47D Human Breast Cancer Cells Total RNA isolated from cells that had been treated with vehicle alone (lanes l-6) or 100 nM MPA (lanes 7-l 1) for 0 h (lane 1) 1 h (lanes 2 and 7) 3 h (lanes 3 and 8) 6 h (lanes 4 and 9) 12 h (lanes 5 and lo), or 24 h (lanes 6 and 11) was subjected to Northern blot analysis. The autoradiograms from the resulting blot hybridized with the c-jun cDNA (upper panel) or the PRA/calcyclin cDNA (middle panel) are shown. The hybridization signals were quantitated, and the data, corrected for gel loading, have been expressed relative to untreated control values, each arbitrarily given a value of 1. Each separate experiment had its own Individual control, which was arbttrarily give a value of 1. The results are presented in the lower panel as the mean k SEM of one experiment for the 0.5 and 48 h points, three separate experiments for the 1 h point, and five separate experiments for 3, 6, 12, and 24 h points.

c-jun

PRA

Fig. 2. Effects of Various Concentrations of MPA on c-jun mRNA Level in T-47D Human Breast Cancer Cells Total RNA isolated from cells that had been treated for 6 h with vehicle alone (lane l), 0.1 nM MPA (lane 2), 1 nM MPA (lane 3) 10 nM MPA (lane 4), 100 nM MPA (lane 5) and 1000 nM MPA (lane 6) was analyzed by Northern blotting. The top panel shows the hybridization pattern obtained using the c-jun cDNA, and the bottom panel shows that obtained with the PRA/calcyclin cDNA.

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Progestin Regulation of jun in T-47D Cells

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the protein synthesis inhibitor. These data suggest that the effect of progestin on the c-jun mRNA level is direct and does not require ongoing protein synthesis.

c-jun

Effects of MPA on Other Members of Genes

PRA

12

3

4

5 B

c-jun

C-jun belongs to a multigene family, of which two other members, jun-B and jun-D, have been identified (17). Since complex interactions may occur between members of this family, we have also investigated the effects of progestins on their expression. The response of junB mRNA levels to MPA was biphasic. MPA was found to decrease jun-B mRNA levels 1 h after treatment in T-47D cells (Fig. 5). Thereafter, @n-B mRNA levels increased and became elevated 12, 24, and 48 h after treatment compared to the untreated control values. MPA had little, if any, effect on @n-D mRNA levels in T47D cells (data not shown). Effects of ICI 164364 Expression

PRA Fig. 3. Effects of Various Compounds on c-jun mRNA Levels in T-47D Human Breast Cancer Cells A, Total RNA isolated from cells that had been treated for 3 h with vehicle alone (lane l), 10 nM MPA (lane 2), 100 nrv progesterone (lane 3), 100 nM dexamethasone (lane 4) 100 nM RU 486 (lane 5) 10 nM R5020 (lane 6) 100 nM all-transretinoic acid (lane 7) 100 nM 17P-estradiol (lane 8) and 100 nM monohydroxytamoxifen (lane 9) was subjected to Northern blot analysis. The patterns of hybridization obtained with the c-jun and PRA/calcyclin cDNAs are indicated. B, Total RNA isolated from cells that had been treated for 3 h with 10 nM MPA alone (lane l), 10 nM MPA plus 10 nM RU 486 (lane 2) 10 nM MPA plus 100 nM RU 486 (lane 3) 10 nM MPA plus 1000 nM RU 486 (lane 4) and 100 nM ICI 164 384 alone (lane 5) was subjected to Northern blot analysis. The patterns of hybridization obtained with the c-jun and PRA/calcyclin cDNAs are indicated.

tions of cycloheximide similar to those used in these experiments, but with only l-h pretreatment (2-h pretreatment was used in the present experiments), have been shown previously under similar conditions to reduce protein synthesis by at least 94% and 90% in MCF-7 and T-47D human breast cancer cells, respectively (1516). Without cycloheximide treatment, results similar to those shown in Fig. 1 were obtained, and the expected 4.5-fold increase in c-iun mRNA was observed (Fig. 4A). A comparison of lane 1 in Fig. 4A with lane 1 in Fig. 48 demonstrates the expected superinduction of c-iun mRNA levels due to cycloheximide treatment alone. However, a further 2.4 f 0.5fold (mean + SEM; n = 3) increase in the c-jun mRNA level was observed after MPA treatment (Fig. 4B). The time course of MPA induction of c-jun mFiNA in the presence of cycloheximide was similar to that in the absence of

of the jun Family

on c-jun, jun-B,

and jun-D

To determine whether alteration of expression of the jun family was a general phenomenon associated with growth inhibition in T-47D cells, the effect of the antiestrogen ICI 164384 on c-jun mRNA was studied. Although ICI 164384 inhibited the growth of these cells (data not shown), the antiestrogen had little, if any, effect on c-jun, jun-B, and jun-I3 mRNA levels (data not shown). Effects of MPA on c-jun Protein Endogenous AP-1 Activity

Levels

and

To determine the functional relevance of MPA regulation of c-jun mRNA, crude nuclear extracts were prepared from T-47D cells at various times after MPA treatment, and the c-jun protein level was visualized by Western blotting and immunodetection. A protein of approximately 39 kilodaltons (kDa) mol wt, detected by the c-jun antibody (Ab-1), was increased by MPA treatment (Fig. 6A). Preadsorption of the c-jun antibody with the synthetic peptide antigen resulted in loss of detection of the p39 protein (data not shown), and normal rabbit immunoglobulin G did not detect this protein (data not shown). A similar result was obtained using another c-jun antibody (Ab-2) which was raised against a synthetic peptide representing amino acids 73-87 in the N-terminal region of v-jun. Interestingly, another .unknown protein of approximately 115 kDa was also detected by Ab-1 , and since the level of this protein did not change after MPA treatment, it was used as a loading control for the Western blots (Fig. 6). An increase in c-jun p39 protein was observed after 3 h of treatment with 100 nM MPA, and the protein remained elevated for up to at least 24 h after treatment. The average fold increase in c-jun protein 24 h after treatment was 3.8-fold (range, 3.2- to 4.4 fold; n = 2) above the control value. Neither 100 nM dexamethasone nor dihydrotestosterone was able to increase c-jun under

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Fig. 4. Effect of Cycloheximlde

Treatment on the Ability of MPA to Increase c-jun mRNA Level in T-47D Human Breast Cancer Cells A, Total RNA isolated from untreated cells (lane 1) and cells treated with 100 nM MPA for 0.5 h (lane 2), 1 h (lane 3) 3 h (lane to Northern blot analysis. The patterns of hybridization 4)) 6 h (lane 5) 12 h (lane 6) 24 h (lane 7) and 48 h (lane 8) was subjected obtained with the c-iun and PRA/calcyclin cDNAs are indicated. For each separate experiment the hybridization signals were quantitated, and the data, corrected for gel loading, expressed relative to the untreated control value in each experiment, arbitrarily given a value of 1. These data are shown as histograms in the bottom panel. B, Total RNA was isolated from cells that had been pretreated for 2 h with 50 PM cycloheximide and then treated with vehicle alone (lane 1) or 100 nM MPA for 0.5 h (lane 2) 1 h (lane 3) 3 h (lane 4)) 6 h (lane 5) 12 h (lane 6) 24 h (lane 7) and 48 h (lane 8). The patterns of hybridization of the resulting Northern blot obtalned with the C-jun and PRA/calcyclin cDNAs are indicated. For each separate experiment the hybridization signals were quantitated, and the data, corrected for gel loading, are expressed relative to the untreated control value in each experiment, arbitrarily given a value of 1. The mean + SEM of three separate experiments are shown as histograms in the bottom pane/.

conditions (Fig. 6B). Moreover, the antiprogestin RU 486 at a IO-fold higher concentration inhibited the MPA-induced increase in the c-jun protein (Fig. 6B). The effect of MPA treatment on endogenous AP-1 activity in T-47D cells was measured by transiently transfecting the cells with a chloramphenicol acetyltransferase (CAT) reporter gene, which was under the control of five 12-0-tetradecanoylphorbol-13-acetate (TPA) response elements (TRE). MPA treatment of these cells resulted in decreased CAT activity (Fig. 7A), which was apparent as early as 6 h after treatment. The maximum decrease obtained was 55% at 16 h, but the activity remained decreased for at least 40 h after treatment. This result was obtained regardless of whether CAT activity was determined per U protein or /I-galactosidase activity. As expected, treatment of the transiently transfected cells with the phorbol ester TPA increased CAT activity (Fig. 7A). Concurrent treatment of the cells with MPA and the antiprogestin RU 486 resulted in a reversal of the MPA effect on CAT activity (Fig. 7B). These data suggest that although MPA increased the expression of c-jun and jun-B, the overall effect of the progestin was to decrease the apparent endogenous AP-1 activity under these growth condithese

tions.

DISCUSSION C-jun expression is increased not only by growth stimulatory signals (9), but also in some cases by growth

inhibitory and terminal differentiation signals (9). Although c-jun obviously plays an important part(s) in mediating growth modulatory signals, the exact role of this gene is unclear. It is known that c-jun encodes a protein that forms heterodimers with the product of the c-fos gene. These dimers form the transcription complexes that bind to AP-1 sequences (9). However, c-jun can form homodimers with itself as well as heterodimers with other members of the jun family (9, 17) and other members of the fos family of genes (9, 17). Moreover, there are a number of other factors that can modulate the activity of AP-1 -like complexes (18). More recently, data supporting an interaction of the components comprising AP-1 complexes with members of the steroid hormone receptor family have been reported (10-13). Steroid hormone receptors and the AP-1 complex comprise two distinct classes of transcription factors that can interact, possibly directly, to modulate each other’s activity (9-13). Furthermore, it has also been shown that estrogens, 1 a,25dihydroxyvitamin DSr and glucocorticoids can regulate the transcription of both c-jun and c-fos genes (19-22). The data in this paper add progestins to the list of steroid hormones that can regulate expression of the c-jun gene. Since estrogens have been found to increase the expression of this gene in the rat uterus, it was initially surprising that progestins, growth inhibitory agents under most circumstances in T-47D human breast cancer

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Progestrn

Regulation

1

of jun in T-47D

Cells

2

4

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115 kDa

39 kDa

PRA

B.

115 kDa

6. Effect of MPA and Other Compounds on c-jun Protein Level in T-47D Human Breast Cancer Cells A, Ten micrograms of crude nuclear extract protein isolated from T-47D cells treated with vehicle alone (lane 1) or 100 nM MPA for 3 h (lane 2) 6 h (lane 3) 9 h (lane 4) and 24 h (lane 5) were subjected to Western blotting analysis, as described in Materials and Methods. C-jun Ab-1 was used in this experiment to detect the 39-kDa c-jun protein. The antibody also detected a 115kDa protein that did not change after MPA treatment and, therefore, has been used as a loading control. 8, Ten micrograms of crude nuclear extract protein isolated from T-47D cells treated for 24 h with vehicle alone (lane l), 100 nM MPA (lane 2) 100 nM MPA plus 1000 nM RU 486 (lane 3) 100 nM dexamethasone (lane 4) and 100 nM dihydrotestosterone (lane 5) were subjected to Western blotting analysis, as described in Materials and Methods. C-jun Ab-1 was used in this experiment to detect the 39-kDa c-jun protein. The antibody also detected a 115kDa protein that did not change due to MPA treatment and, therefore, has been used as a loading control. Fig.

0

1

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TIME (hrs) 5. Effect of MPA on jun-B mRNA Level in T-47D Human Breast Cancer Cells Total RNA isolated from cells that had been treated with vehicle alone (lanes 1) or 100 nM MPA for 1 h (lanes 2) 3 h (lanes 3) 6 h (lanes 4) 12 h (lanes 5) or 24 h (lanes 6) was subjected to Northern blot analysis. The autoradiograms from the resulting blot hybridized with thejun-B cDNA (upperpanel) or the PRA/calcyclin cDNA (middle panel) are shown. For each separate experiment the hybridization signals were quantitated, and the data, corrected for gel loading, are expressed relative to the untreated control value in each experiment, arbitrarily given a value of 1. The results are presented in the lower panel as the mean + SD of two separate experiments for the 1 h point and the mean + SEM of three separate experiments for the 3, 6, 12, and 24 h points and one for the 48 h point. Please note that the SD for the 1 h point is 0.005 and is not resolved from the mean value in the program used to draw the histograms (Cricket Graph 1.3).

Fig.

cells, increase the expression of this gene. However, in the chick oviduct, estrogen decreased c-@n expression (20) while tamoxifen rapidly increased c-jun mRNA levels (23). Furthermore, tumor necrosis factor-a, a potent antimitotic factor for endothelial cells, caused an increase in c-jun mRNA levels in these cells (24). As well, c-jun expression is increased by TGFP in A549 lung adenocarcinoma cells when the end result of TGFP stimulation is growth inhibition (25). TGFP also increased c-jun expression in K562 human erythroleukemia cells, although it had no effect on their proliferation, while in AKR-2B mouse fibroblast cells, although TGFP stimulated their growth, no effect on c-jun expression was found (25). Obviously, the regulation of c-jun

expression can often be a point of convergence of major growth modulatory signals generally. The end result of that signal is determined by the genetic program of each individual cell type. With respect to human breast cancer cells, the phorbol ester TPA, which is known to increase AP-1 activity and increase the transcription of c-jun (26), has been found to be growth inhibitory for human breast cancer cells in culture (27). This suggested that increased AP1 activity was associated with growth inhibition in this system. In contrast, when the proliferation of MCFJ cells was stimulated by epidermal growth factor, or TGFa increased c-fos expression was observed (28) and increased AP-1 activity was shown to be associated with the synergistic stimulation of proliferation by insulin and estrogen in MCF-7 cells (29). Regulation of AP-1 activity, however, is complex, and other factors, including other members of thejun and fos families, are thought to effect AP-1 activity. It is possible, therefore, that regulation of these other factors may contribute to an overall growth stimulatory or inhibitory outcome. With this in mind, we examined the effect of progestins

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Fig. 7. Effect of MPA Treatment on Overall Endogenous AP1 Activity in T-47D Human Breast Cancer Cells A, T47D cells (2 x 10”) were transiently transfected with 5 pg 5 x TRE-tk-CAT and 3 pg pCHll0 and treated with vehicle alone or 100 nM MPA or 100 nM TPA. The cells were harvested at the times indicated, supernatants were prepared, and CAT and @-galactosidase activities were measured as described in Materials and Methods. Results of three separate experiments (mean + SEM) are expressed as fold induction above the vehicle control value, arbitrarily given a value of 1. Any points that do not have error bars represent values for which the error bars are within the dimensions of the symbol used. B, T47D cells (2 x 106) were transiently transfected with 5 pg 5 x TRE-tk-CAT and 3 pg pCHll0 and treated for 16 h with the indicated concentrations of MPA or 100 nM MPA plus 1000 nM RU 486. The cells were harvested, supernatants were prepared, and CAT and @-galactosidase activities were measured as described in Materials and Methods. The results of two separate experiments (mean & SD are expressed as the fold induction above the vehicle control value, arbitrarily given a value of 1.

on other members of thejun family. &n-D mRNA were unaffected by progestin treatment, but mRNA levels were initially decreased, with peak

levels jun-B

inhibition occurring 1 h after treatment. Thereafter, jun-B mRNA levels increased and were higher than control

values 12-48 h after treatment. Furthermore, c-fos mRNA levels are also transiently increased by progestins in T-47D cells, with a maximum stimulation of c-fos mRNA occurring 30 min after treatment (30). We have confirmed this initial progestin-induced increase in c-fos mRNA in T-47D cells, but our data also indicate that a second increase in c-fos mRNA occurs 24 h after treatment (manuscript in preparation). The above observations support the conclusion that several members of the gene families that contribute to AP-1 complexes are regulated by progestins in T-47D cells. Therefore, a number of interactions between the members of these gene families could be occurring. In an attempt to understand the overall biological effect of progestin on AP-1 expression, we measured AP-1 activity in MPA-treated T-47D cells by activation of transiently transfected 5 x TRE-tk-CAT. The results indicated that overall AP-1 activity was decreased by progestin treatment. However, it is unclear at this stage what is the mechanism of this inhibition. Since the effect is maximal at 16 h after treatment, the increased jun-B expression may be responsible for the inhibitory action (31). Alternatively, activated human progesterone receptors have been shown to inhibit c-@n-induced 5 x TRE-tk-CAT transcription by a mechanism that does not involve receptor interaction with the 5 x TRE-tkCAT (11). Either mechanism or a combination of both could explain the result obtained. The relatively small effect of TPA on 5 x TRE-tk-CAT activity in T-47D cells compared to other systems may be due to the observation that T-47D cells are much less sensitive to TPA with respect to growth inhibition and other parameters than other breast cancer cell lines (27). Alternatively, T47D cells may have a relatively high basal level of endogenous AP-1 activity compared to other cell lines, which may limit the relative increase that can be measured under the conditions of these experiments. The effect of MPA on c-jun expression in T-47D cells appears to be direct and does not require ongoing protein

synthesis.

Cycloheximide

treatment

alone

caused a superinduction of c-jun mRNA, an effect possibly due to increased stability of the c-jun mRNA (17). A further increase in c-jun mRNA was observed with MPA treatment. The approximately 2-fold increase is less than that seen with MPA treatment alone (i.e. 5.4fold increase) and is, therefore, in contrast to the effects of the same treatment on EGF mRNA levels in T-47D cells (32). In this case, combined MPA and cycloheximide treatment resulted in a greater than 30-fold increase in EGF mRNA steady state levels, while the effect of MPA alone is only an 1 l-fold increase. A possible reason for the difference may reside in the observation that AP-1 complexes autoregulate c-jun expression and cycloheximide would inhibit the synthesis of the rapidly turning over proteins that form AP-1 complexes (9, 17, 26). Another possibility is that there is a maximum level of c-jun transcription, and progestins can only increase it to that level; therefore, relative induction would depend upon the basal level of transcription at the time of treatment. As well as increasing

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Progestin

Regulation

of jun in T-47D

Cells

the steady state levels of c-jun mRNA 5.4-fold, progestins increased the p39 c-jun protein 3.8-fold. The slightly lesser effect on the c-jun protein compared to the c-jun mRNA has been found previously (9, 26), although the reason for this is as yet unclear. The effect of progestin on the c-jun protein, however, seems to be more prolonged than its effect on c-jun mRNA. This suggests that progestin may also affect the stability of the c-jun protein or, alternatively, affect translation rates of the c-jun mRNA (33). With respect to this latter possibility, it is interesting that the c-jun 5’-untranslated region (448 to 453) (34) contains an AGAAGA hexamer that has been previously implicated in steroid hormone regulation of translation (33). In summary, the data presented show that progestins specifically alter the expression and activity of components that can form AP-1 transcription complexes in T47D cells.

MATERIALS

AND METHODS

Materials MPA, progesterone, dexamethasone, retinoic acid, and 17pestradiol were purchased from Sigma (St. Louis, MO). [3”P] Deoxy-CTP was purchased from NEN (Lachire, Quebec, Canada). 4-Hydroxytamoxifen and ICI 164 384 were gifts from ICI (Macclesfield, Cheshire, United Kingdom). RU 486 was a gift from Roussel-UCLAF (Romainville, France). Dulbecco’s Minimum Essential Medium (DMEM) powder was purchased from Gibco/BRL (Burlington, Ontario, Canada), and fetal bovine serum was purchased from UBI (Lake Placid, NY). All other cell culture medium ingredients were purchased from Flow Laboratories (Mississauga, Ontario, Canada).

Cells The cell lines were obtained from sources described previously (4, 5). The cells were grown in DMEM supplemented with 5% fetal bovine serum, glutamine, glucose, and penicillin/streptomycin (4, 5). Cells were plated at 1 x 1 O6 in 11 O-mm dishes and 2 days later treated as indicated in the text. The cells were harvested at the times indicated by scraping with a rubber policeman. After centrifugation, the cell pellet was frozen and stored at -70 C until RNA was isolated. For cycloheximide studies, cells were treated with 50 PM cycloheximide (15, 16) for 2 h before and during MPA or vehicle alone treatment. RNA Extraction

and Northern

Blot

Analysis

RNA was isolated by the guanidinium thiocyanate/cesium chloride method (35). Thirty to 40 pg total RNA were denatured in 50% (vol/vol) formamide and 2.2 M formaldehyde, sizeseparated by electrophoresis on 1% (wt/vol) agarose gels containing 2.2 M formaldehyde, and then blotted onto nitrocellulose (36). Filters were baked for 2 h at 80 C under vacuum and then prehybridized in hybridization solution for at least 3 h. The filters were hybridized sequentially with human c-jun, the human jun-B cDNA probes (both kindly provided by Dr. M. Karin), and the mouse jun-D cDNA probe (American Type Culture Collection, Rockville, MD). Hybridizations, usually for 48 h, were performed at 42 C in the presence of 50% (vol/vol) deionized formamide, 5 x Denhardt’s solution [l x Denhardt’s = 0.02% (wt/vol each of BSA, Ficoll, and polyvinylpyrrolidine], 5 x SSPE (1 x SSPE = 1.15 M NaCI, 0.01 M NaH,PO,, and

1631

1 mM EDTA), 250 Kg/ml denatured salmon sperm DNA, and 0.1% sodium dodecyl sulfate (SDS). At the end of the hybridization period, the blots were washed twice in 2 x SSC-0.1% SDS (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate) for 15-30 min at room temperature, followed by three 20-min washes in 0.1 x SSC-0.1% SDS at 65 C. Filters were also hybridized with the human PRA/calcyclin cDNA (14) as a control for differences in the amount of RNA loaded on the gel. The level of PRA/calcyclin mRNA is unaffected by MPA treatment under the conditions used in this study. Filters were exposed to Kodak XAR film (Eastman Kodak, Rochester, NY) at -70 C with an intensifying screen. Quantitation was achieved by scanning the autoradiograms using a Hewlett-Packard Scan Jet 2 flat-bed scanner (Santa Clara, CA), and the data were analyzed using the Image program. Transfection

and

CAT Assays

T-47D cells (2 x lo6 cells/lOO-mm diameter dish) were transiently transfected using the calcium phosphate/glycerol shock method (37) with modifications. After 6-h incubation with the calcium phosphate-precipitated DNA, the cells were shocked with 20% (vol/vol) glycerol-DMEM for 2.5 min. The cells were then treated with vehicle alone or 100 nM MPA, as indicated. At the times indicated, cells were harvested, and a supernatant fraction was prepared as previously described (38). The CAT and @-galactosidase activities of an aliquot of each supernatant were measured using standard protocols (38, 39). The cells were transfected with 5 pg 5 x TRE-tk-CAT (40) (kindly provided by Dr. M. Karin), 3 fig pCH110 (j3-galactosidase expression vector, Pharmacia, Baie d’llrfe, Quebec, Canada). Western

Blot Analysis

All steps were carried out at 4 C. T-47D cells were homogenized in 5 mM KCI, 0.5 mM MgC12, 25 mM Tris-Cl (pH 7.5) 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride, and a crude nuclear pellet was collected by centrifugation at 800 x g for 10 min. This pellet was washed in the above buffer containing 0.1 M sucrose. The nuclear pellet was extracted for 60 min with 1.2 M NaCI, 0.1% (vol/vol) Nonidet P-40, 25 mM Tris-Cl (pH 7.5) 1 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride, and then centrifuged for 60 min at 100,000 x g. The resulting supernatant was sonicated, and the protein was determined by the Lowry method (41). An aliquot of the nuclear extract containing 10 gg protein was added to an equal volume of loading buffer [0.065 M Tris-Cl (pH 6.8) 4% (wt/vol) SDS, 10% (vol/vol) glycerol, 1.43 M 2@mercaptoethanol, and 0.00125% (wt/vol) bromophenol blue], boiled for 10 min, separated on a 7.5% SDS-polyacrylamide gel, and transferred to nitrocellulose. After blocking the membrane with 1% (wt/vol) nonfat milk in Tris-buffered saline containing 0.5% Tween-20 (TTBS), the blots were incubated for 1 h at room temperature, and the membrane was rinsed three times with TTBS and then incubated for 1 h with 1.5 pg/ ml Ab-1 (c-jun/AP-1 rabbit, affinity-purified polyclonal antibody raised to a synthetic peptide representing amino acids 209225 in the DNA-binding domain in the C-terminal region of vjun; Oncogene Science, Manhasset, NY). After washing in TTBS, the membrane was incubated for 1 h with a 1:3,000 dilution of goat antirabbit immunoglobulin G conjugated to horseradish peroxidase (Bio-Rad, Mississauga, Ontario, Canada). After washing in TTBS, immunoreactive bands were visualized by incubation with luminol (according to manufacturer’s instructions; ECL Western blotting detection system Amersham, Oakville, Ontario, Canada) and exposure to Hyperfilm-ECL (Amersham). C-jun antibody Ab-2 (Oncogene Science) was also used at 1.5 bg/ml.

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MOL 1632

ENDO.

1992

Acknowledgments

Received February 27, 1992. Revision received July 27, 1992. Accepted July 27, 1992. Address requests for reprints to: Dr. Leigh C. Murphy, Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3. This work was supported by the Medical Research Council of Canada, the NCI of Canada, and equipment grants from the H. E. Sellers Foundation and the Canadian Women’s Breast Cancer Foundation. Supported by a Manitoba Health Research Council postdoctoral fellowship. T NCI (Canada) scientist. l

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