Effect of Overexpression of Progesterone Receptor A on Endogenous Progestin-Sensitive Endpoints in Breast Cancer Cells

Eileen M. McGowan and Christine L. Clarke

Westmead Institute for Cancer Research University of Sydney Westmead Hospital Westmead, New South Wales 2145, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, which differ in the N-terminal region and exhibit different activities in vitro, with PRA demonstrating dominant negative inhibitory effects on the activity of PRB and other nuclear receptors. PRA and PRB are expressed in target tissues at comparable levels although cells expressing a predominance of one isoform can be identified. In breast cancers, PRA is expressed at high levels in some tumors, and this may be associated with features of poorer prognosis. To investigate the role of PRA overexpression in PR-positive target cells, the effect of PRA induction on cell proliferation and expression of endogenous progestin-sensitive genes, SOX4 and fatty acid synthetase (FAS), was examined using PR-positive T-47D cell lines, which express a predominance of PRB, in which PRA could be increased 2- to 20-fold over basal levels. No effect of PRA induction was noted on cell proliferation, but marked changes in morphology, consistent with loss of adherent properties, were observed. Increases up to 4-fold in the relative PRA levels augmented progestin induction of SOX4 mRNA expression, and RU486 treatment revealed a progestin agonist effect. There was no consistent effect of PRA induction on progestin-mediated increases in FAS mRNA levels under these conditions. Clones with PRA:PRB ratios greater than 15 were associated with diminished progestin responses on both SOX4 and FAS mRNA expression. These data show that PRA overexpression is associated with alteration in adhesive properties in breast cancer cells and effects on endogenous progestin targets that were dependent on the cellular ratio of PRA:PRB. The results of this study are consistent with the view that PRA expression can fluctuate within a broad range in target cells without influencing the nature of progestin action on downstream targets, but that overexpression of PRA, such as is seen in a proportion of breast cancers, may be associated with inhibition of progestin action and features of poor prognosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The steroid hormone progesterone plays a pivotal role in normal female development and reproduction (1) through its interaction with the nuclear progesterone receptor (PR). The expression of human PR is controlled by two promoters (2) that direct the synthesis of mRNA transcripts encoding two receptor proteins, PRB and PRA, in breast cancer cells (3). The two proteins are identical except that the shorter A protein is N-terminally truncated by 164 amino acids. PRA and PRB are coexpressed in the same target cells in the human (4), and their relative expression, where it has been examined, generally is close to unity (1) although cells expressing a predominance of one of the isoforms are observed in target tissues under some physiological circumstances (4). PRA is the predominant isoform in the rodent (1) and is widely expressed in the macaque reproductive system (5) and in the human uterus (4, 6). In breast cancers, high levels of PRA can occur: previous studies using immunoblot analysis documented very high levels of PRA (up to 100 fold higher than PRB) in a subset of breast tumors (7). Transgenic mice overexpressing PRA exhibited features in their mammary glands that were abnormal and commonly associated with neoplasia (8), and in vitro studies have suggested that poorly differentiated endometrial cancer cells express only PRA (9).

The PRA and B proteins have different capacities to activate target genes, and regions of the PR protein to which these different effects can be attributed have been identified (10, 11, 12). Insights into the different transcriptional activities of PRA and PRB have been obtained by transient cotransfection of PRA and/or PRB and reporter constructs containing progestin-responsive sequences ranging from the simple PRE-tk-chloramphenicol acetyl transferase (CAT) [containing one copy of a palindromic progesterone responsive element (PRE)] to more complex constructs such as those incorporating the mouse mammary tumor virus long terminal repeat (MMTV-LTR), which contains multiple hormone-responsive elements, into a variety of cell lines (13, 14, 15, 16, 17, 18, 19). In all cell types examined, PRB exhibited hormone-dependent transactivation irrespective of the complexity of the response elements, whereas the transcriptional activity of PRA was cell- and reporter specific. Interestingly, PRA acted as a transdominant inhibitor of PRB where PRA had little or no transactivational activity (14, 18) and, moreover, PRA regulated the transcriptional activity of other nuclear receptors such as glucocorticoid, mineralocorticoid, androgen, and estrogen (14, 15, 18, 20, 21), suggesting that PRA may play a central role in regulation of activity of a number of nuclear receptors in addition to PRB.

The different transcriptional activities of PRA and B, and the inhibitory activity of PRA in vitro, suggest that tissues that express different relative levels of the two proteins, and in particular high levels of PRA, may have impaired responsiveness to progesterone and other nuclear receptor ligands. However, while transient transfection studies have yielded important insights into the mechanisms of PRA and PRB transactivation, most have been carried out in cells that are not normally progestin targets, and the endpoints examined have consisted of hormone-responsive elements linked to reporter sequences. Consequently, little is known of the effect of altering the ratio of PRA and PRB in PR-positive cells, in particular on endogenous targets of progesterone action. In this study, to examine the significance of overexpression of PRA on endogenous progestin-responsive targets, PR positive T-47D breast cancer cell lines have been constructed in which PRA can be induced 2- to 20-fold, allowing manipulation of PRA levels and consequently of the ratio of PRA:PRB in favor of PRA. The consequence of PRA overexpression on endogenous progestin-sensitive endpoints has been examined, including cell proliferation, cell morphology, and expression of SOX4, which is directly transcriptionally regulated by progestin (22) and the progestin-sensitive gene fatty acid synthetase (FAS) (23).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction and Sequence Verification of the pOP13 hPRA Plasmid
The pOP13 hPRA plasmid was sequenced in both sense and antisense directions using PRA- and pOP13-specific primers. Three nucleotide base changes were observed within the hPRA coding sequence, which differed from the published sequence (GenBank accession X51730). A change from A to G (AGC = GGC) at position 2130 was noted, which would result in an amino acid change from serine to glycine. A change from TGT to GTC at positions 2220–2222 was noted, which would result in an amino acid change from cysteine to valine. Amplification of these regions in genomic DNA indicated that the sequences observed in the plasmid were identical to those observed in genomic DNA but differed from the published sequence. A change from C to T at position 2633, resulting in a silent base change, was observed in the pOP13 hPRA plasmid and was present in pSG5-hPR1, but not in genomic DNA or the published sequence.

Characterization of T-47DhPRA Cell Lines
After sequence verification, the pOP13-hPRA plasmid was used to construct stable transfectants of two breast cancer cell lines: T-47D cells, which contain both PRA and PRB, with PRB predominating, and an ER-positive, PR-negative clone of MCF-7M cells, MCF-7M11 cells, in which PR was undetectable by Northern blot and immunoblot analysis. T-47D cells were chosen to construct cell lines in which PRA levels could be manipulated, as they are PR positive and highly progestin responsive and have been used extensively in published studies as a model to examine PR action. A PR-negative MCF-7 clone was used to constitutively express PRA, to characterize the activity of PRA encoded by the pOP13-hPRA plasmid, in the absence of endogenous PRA or PRB, but in a context, when PR is expressed (as in the wild-type MCF-7 cells), where progestin response is normally seen.

T-47D clonal cell lines constitutively expressing the lac repressor protein predominantly expressed PRB (PRA:PRB ratio, 0.56–0.89), and the ratio of the two PR isoforms was stable in these lines upon passage (data not shown). Transfection of the pOP13-hPRA plasmid into these clonal cell lines yielded 18 cell lines (T-47DhPRA cells): 5 of these were examined further (Fig. 1Go; T-47DhPRAN3, -N4, -N5, and -E3) and T-47DhPRAB8 (not shown). The basal level of PR expression and the relative expression of PRA and PRB were similar in T-47DhPRA cells and in the parent lines ( Figs. 6–8GoGoGo and data not shown), indicating little expression of PRA from the pOP13-hPRA plasmid in the absence of induction using isopropyl-ß-thiogalactosidase (IPTG), except in the case of the N5 (Fig. 1Go) and B8 (not shown) cell lines, which displayed basal levels of PRA greater than PRB, suggesting incomplete suppression of the pOP13-hPRA plasmid in these cell lines. Induction of PRA expression using IPTG, which sequestered the lac repressor and allowed transcription from the pOP13-hPRA plasmid, resulted in induction of PRA over basal levels (Fig. 1Go): this caused a marked change in the ratio of PRA:PRB, in favor of PRA (PRA:PRB ratio ranged from 2–20 in all the cell lines examined).



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Figure 1. Characterization of PRA and PRB Expression in T-47DhPRA Cell Lines

T-47DhPRA cell lines were constructed using the Lac-Switch inducible mammalian expression system as described in Materials and Methods. The figure shows immunoblot analysis (30 µg total protein/lane) of T-47DhPRAN3, -N4, -N5, and -E3 cells stably expressing both the p3'SS and the pOP13 hPRA plasmids. Cells were treated with IPTG (+; 10 mM) or vehicle (-). PRA:PRB ratios were calculated after quantitative densitometry.

 


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Figure 6. Progestin Effect on SOX4 mRNA Expression in T-47DhPRA Cells

T-47DhPRA cells were treated with IPTG (10 mM) for 24 h before treating with ORG2058 (10 nM). Cells were harvested 24 h later and total RNA and cytosol protein prepared. A, Northern blots (30 µg RNA/lane) were probed with SOX4 cDNA and then stripped and reprobed for 18S rRNA. PRA and B protein were visualized on immunoblots (25 µg cytosol protein/lane). B, Histogram of SOX4 expression (AU) after correcting for loading: hatched bars, -IPTG; open bars, +IPTG. PRA:PRB ratio is indicated above the bars. C, Squares, triangles, diamonds, and crosses show the results from four separate experiments.

 


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Figure 7. Progestin Effect on Endogenous FAS mRNA in T-47DhPRA

RNA and protein were run on Northern and immunoblots and probed for SOX4 mRNA and PR protein as described in the legend to Fig. 6Go. A, The figure is a composite of two blots, one blot containing the data for T-57D-N, T-47DhPRAN4 cells and the other blot containing the data for T-47DhPRAE3 cells; each blot was derived from a separate experiment. B, Histogram of FAS expression as described in Fig. 6Go. C, Squares, triangles, diamonds, and crosses show the results from four separate experiments.

 


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Figure 8. Progestin and Antiprogestin Effects on Endogenous FAS and SOX4 mRNA in T-47DhPRA Cells

T-47DE parent cells and T-47DhPRAE3 and -N5 cells were treated with IPTG (10 mM) or vehicle for 24 h and then treated with ORG2058 (10 nM), RU486 (100 nM), or their combination. Cells were incubated for a further 24 h before harvesting and preparation of total RNA and cytosol protein. Northern blots (30 µg total RNA/lane) were probed for FAS and SOX4 mRNA and reprobed for ß-actin as a loading control. PRA and B proteins were visualized on immunoblots (25 µg cytosol protein/lane).

 
Time- and Concentration Dependence of PRA Induction in T-47DhPRA Cells
The time- and concentration dependence of PRA induction in T-47DhPRA cells were measured in two T-47DhPRA cell lines, T-47DhPRA-E3 (Fig. 2Go) and T-47DhPRA-N4 (data not shown), and similar results were obtained for each cell line. Increases in PRA protein levels were observed as early as 4 h after IPTG treatment and continued to 48 h (Fig. 2Go) and 72 h (data not shown) thereafter. The increase in PRA protein after IPTG treatment was consistent with increased transcription of mRNA encoding PRA from the pOP13-hPRA plasmid, and no IPTG-mediated increase in endogenous PR transcripts was observed (Fig. 2CGo, inset). There was no difference in PRA induction at the IPTG concentrations tested (Fig. 2BGo), or if the IPTG concentration was increased to 20 mM (data not shown). An increase in basal PRA levels was observed in untreated cells over time (Fig. 2Go, A and B). This was also noted for PRB (data not shown) and has been described previously, probably as a consequence of time in culture (Ref. 24 and our unpublished results).



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Figure 2. Time- and Concentration Dependence of PRA Induction in T-47DhPRAE3 Cells

T-47DhPRAE3 cells were treated with IPTG (1, 5, or 10 mM) or vehicle, 24 h after seeding and cells were harvested 7, 12, 24, 31, and 48 h thereafter. A, Representative immunoblot (30 µg total protein/lane). B, Levels of PRA and PRB in arbitrary units (AU) calculated after quantitative densitometry. Inset shows an immunoblot of PRA and PRB protein at 48 h. {square}, Vehicle; {circ}, 1 mM IPTG; {diamond}, 5 mM IPTG; {triangleup}, 10 mM IPTG. C, Total RNA was isolated 24 h and 48 h after IPTG treatment and PR mRNA levels detected by Northern blot (20 µg/lane). Arrow indicates the major 11.4-kb endogenous PR transcript; pOP13hPRA indicates the position of the PRA transcript encoded by the plasmid. Inset shows fold induction of pOP13-hPRA transcript (open bars) compared with the predominant 11.4-kb PR mRNA transcript (stippled bars) after correction for loading. The data are representative of two experiments in separate T-47DhPRA clones in which the same results were observed.

 
Progestin Effect on Cell Proliferation and Morphology in T-47DhPRA Cells
The doubling time of T-47DhPRA cells stably expressing the pOP13-hPRA plasmid was similar to that of the parent cells expressing only the plasmid encoding the lac repressor (Fig. 3Go) and to that of wild-type T-47D cells (data not shown). Induction of PRA had no effect on cell proliferation (Fig. 3Go, compare circles and diamonds) in the absence of the progestin ligand. Treatment with progestin resulted in growth inhibition in the parent T-47DE and in the T-47DhPRAE3 cells, and there was no difference in the effect seen in cells in which PRA had been induced by IPTG (Fig. 3AGo, compare squares and triangles), when cell proliferation was measured using an assay based on cell viability. However, when cell counts were determined, it was noted that induction of PRA resulted in a greater loss of cells from the monolayer than that observed in the absence of PRA induction (Fig. 3BGo, T-47DhPRAE3 cells, compare squares with triangles). This effect was noted consistently in four additional T-47DhPRA clonal cell lines (data not shown). Prolonged exposure to progestin for longer than 6 days resulted in a more pronounced effect on PRA-overexpressing cells. Cells in which PRA had been induced were more sensitive at these late time points to the growth-inhibitory effects of progestins, and maximal effects that were greater than those seen in uninduced cells were observed (data not shown).



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Figure 3. Progestin Effect on Cell Proliferation in T-47DhPRA Cells

T-47DE and T-47DhPRAE3 cells were seeded in 96-well plates or 25-cm2 flasks at a density of 2 x 104 cells/ml as described in Materials and Methods. Twenty four hours later (day -2) the medium was supplemented with IPTG (10 mM) or vehicle. Cells were treated with ORG2058 (10 nM) or vehicle on day -1. A, Cell proliferation was measured using the CellTiter 96 nonradioactive cell proliferation assay on days -2, -1, 2, 4, and 6. B, Cell number was determined by hemocytometer on days -2, 0, 3, and 5. {circ}, Vehicle; {triangleup}, ORG2058 (10 nM); {diamond}, IPTG (10 mM); {square}, IPTG (10 mM) and ORG2058 (10 nM). All experiments were performed in triplicate and the data expressed as mean ± SEM. Where the error bars are not visible, they did not exceed the size of the symbol.

 
Examination of the morphology of T-47DhPRA cells revealed that progestin treatment of cells in which PRA had been induced caused rounding and detachment of cells from the monolayer (Fig. 4Go, D and F) compared with cells treated with progestin without PRA induction (Fig. 4Go, C and E). Cells that detached from the monolayer retained viability, as evidenced by the assay shown in Fig. 3AGo, and also by their ability to readhere to tissue culture flasks in fresh medium lacking progestin (data not shown). Induction of PRA in the absence of progestin had no effect on cell morphology (Fig. 4Go, compare A and B).



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Figure 4. Effect of Progestin Treatment on Morphology of T47DhPRA Cells

T-47DhPRAE3 cells were seeded in 75-cm2 flasks at a density of 2 x 104 cells/ml and treated with IPTG and ORG2058 as described in the legend to Fig. 3Go. Cells were photographed on days 0, 2, 3, and 4 after ORG2058 treatment. Panels A–D show the morphology of the cells at day 3 (magnification 170x): A, vehicle; B, IPTG (10 mM); C, ORG2058 (10 nM); D, IPTG (10 mM) and ORG2058 (10 nM). E and F, High magnification views, day 4 after ORG2058 treatment: E, ORG2058 (10 nM); F, IPTG (10 mM) and ORG2058 (10 nM) (magnification 340x).

 
Transcriptional Activity of PRA in PR-Negative MCF-7M11 Cells Stably Transfected with the pOP13 hPRA Plasmid
To document the transcriptional activity of the PRA protein encoded by the pOP13-hPRA plasmid, PRA was constitutively expressed by stable expression of this plasmid in PR-negative MCF-7M11 cells, without the lac repressor encoding (p3'SS) plasmid. Activity of the PRA protein encoded by the pOP13- hPRA plasmid was determined by evaluation of its ability to down-regulate estrogen receptor (ER) and its ability to induce the progestin-responsive pMSG-CAT reporter. PRA protein was detected in MCF-7M11 cells after stable expression, with the expected progestin-mediated increase in size being observed (Fig. 5CGo). Progestin treatment of MCF-7M11 hPRA cells resulted in down-regulation of ER mRNA expression (Fig. 5AGo), a previously described effect of progestin on ER expression (25), and increased activity of the transiently transfected pMSG-CAT plasmid (Fig. 5BGo), demonstrating the activity of PRA encoded by the pOP13-hPRA plasmid. PR protein was absent in MCF-7M11 parent cells, and there was no effect of progestin treatment on either ER mRNA levels or on pMSG-CAT activity in these cells (Fig. 5Go).



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Figure 5. Transcriptional Activity of PRA Encoded by pOP13- hPRA Plasmid

MCF-7M11 and MCF-7M11 cells stably transfected with pOP13 hPRA (MCF-7M11 hPRA cells) were treated with ORG2058 (10 nM) (+) or vehicle (-) for 24 h before harvesting and preparation of total RNA and protein. A, Northern blots (30 µg total RNA/lane) hybridized with a cDNA probe for ER. Blots were stripped and reprobed for 18S rRNA. B, PR protein was visualized on immunoblots (25 µg cytosol protein lane). C, MCF-7M11 and MCF-7M11 hPRA cells were transiently transfected with pMSG-CAT and CAT activity measured after treatment (24 h) with ORG2058 (10 nM) or vehicle. Data were normalized for transfection efficiency by cotransfection with a plasmid encoding ß-galactosidase, pCH110. Experiments were performed in triplicate and the data expressed as the mean fold induction ± SEM.

 
Effects of Increased hPRA Expression on Endogenous Progestin-Responsive Genes in T-47DhPRA Cells
To determine whether alteration in the ratio of PRA:PRB, in favor of PRA, influenced expression of endogenous progestin-sensitive genes, T-47DhPRA cells were treated with progestin, with or without PRA induction, and the expression of SOX4 (Fig. 6Go) and FAS (Fig. 7Go) mRNA was measured. Addition of IPTG increased PRA levels in the T-47DhPRA cells but not in the T-47DN parent cell line (Fig. 6AGo). The magnitude of the increase in PRA levels ranged from 2-fold in the -E3 cells to 3.3-fold in the N5 cells (Fig. 6AGo, lower panel). PR protein levels were decreased in progestin-treated cells, including T-47DhPRA cells in which PRA had been induced by IPTG, consistent with the known down-regulatory effect of progestins on PR (26), except in the case of T-47DhPRAN5 cells, in which persistence and even accumulation of PRA was observed upon progestin treatment (Fig. 6AGo, lower panel).

The effect of PRA induction on the endogenous progestin-responsive gene SOX4 was evaluated by examining the same samples of T-47DhPRA cells for SOX4 mRNA expression. SOX4 mRNA was increased by progestin treatment in the parent T-47DN cells and in the T-47DhPRAE3, -N5 (Fig. 6Go), and -N4 (data not shown) cell lines. Induction of PRA augmented the induction of SOX4 mRNA expression in T-47DhPRAE3 and -N5 (Fig. 6Go, A and B) and -N4 cells (data not shown). There was no effect of PRA induction on the parent N cells (Fig. 6Go, A and B). The augmentation of the progestin-mediated effect was proportional to cellular levels of PRA (r2 = 0.96, not shown) but not PRB (r2 = 0.28, not shown), with greater increases being observed with higher PRA:PRB ratios (Fig. 6BGo). The relationship between PRA:PRB ratio and SOX4 mRNA expression was explored in a number of separate experiments (Fig. 6CGo): these showed that SOX4 mRNA levels were proportional to the ratio of PRA:PRB (P < 0.0001, analysis of covariance) in all cell lines tested (n = 4). The effect of PRA on another endogenous progestin target, FAS, was different from that noted for SOX4. FAS mRNA expression was increased in the T-47DN parent cell line and in the T-47DhPRAE3 and -N4 cells after addition of ORG2058 (Fig. 7Go). Over a range of experiments, where the same RNA shown in Fig. 6CGo was probed for FAS, increased PRA levels had a variable effect on ORG2058 induction of FAS (Fig. 7BGo), suggesting that if there was a relationship between PRA or PRB levels and progestin-mediated increase in FAS mRNA, it was not a consistent one (Fig. 7CGo).

Progestin and Antiprogestin Effects on Endogenous FAS and SOX4 mRNA Expression in T-47DhPRA Cells
The expression of FAS and SOX4 mRNA was determined in T-47DhPRAE3 and -N5 cells and in the parent T-47DhPRAE cell line treated with a combination of ORG2058, RU486, IPTG, and vehicle as shown in Fig. 8Go. Progestin-mediated induction of FAS and SOX4 mRNA was observed in all cells, and induction of PRA with IPTG augmented the progestin induction of SOX4 mRNA, with little effect on FAS mRNA levels, as described in Figs. 6Go and 7Go. RU486 alone had little effect on FAS mRNA levels, with or without PRA induction (Fig. 8Go), whereas SOX mRNA levels were increased in RU486- and IPTG-treated cells in which PRA had been induced, indicating that RU486 displayed some agonist activity on SOX4 expression upon PRA induction (Fig. 8Go, middle panels). Combined treatment with ORG2058 and RU486 resulted in a diminution of the progestin-mediated induction of FAS and SOX4 mRNAs, indicating that RU486 was exerting an antiprogestogenic effect on expression of these genes. The combined effect of ORG2058 and RU486 on FAS mRNA was the same whether or not PRA had been induced (Fig. 8Go, FAS: compare IPTG+ and IPTG- panels). The combination of ORG2058 and RU486 on SOX4 expression was similar to the effect obtained with RU486 alone, whether or not PRA had been induced (Fig. 8Go, SOX4: compare IPTG+ and IPTG- panels).

Effect of Marked PRA Excess on Endogenous Progestin-Dependent Targets
The PRA:PRB ratio observed in the experiments presented in Figs. 6–8GoGoGo ranged from 1–3.3: in these circumstances, PRA induction augmented progestin-mediated increases in SOX4 mRNA expression, with no consistent effect on FAS mRNA expression. To determine the effect of progestin on endogenous targets in cells expressing more marked relative levels of PRA, either in the absence of PRB or with low relative levels of PRB, SOX4 and FAS mRNA levels were determined in MCF-7M11 hPRA cells, which contained only PRA and lacked PRB, and in two clones of the T-47DhPRA cells (N5, B8), which had PRA:PRB ratios of 15 and 20, respectively. Where PRA was the only isoform, as in MCF-7M11 hPRA cells, ORG2058 treatment did not cause an increase in SOX4 or FAS mRNA levels, but instead resulted in a decrease in SOX4 and no effect on FAS mRNA levels (Fig. 9Go).



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Figure 9. Effect of Marked PRA Excess on Endogenous Progestin-Dependent Targets

RNA and protein were isolated from MCF-7M11 hPRA and T-47DhPRA-N5 and -B8 cells and analyzed on Northern blots (30 µg RNA/lane) and immunoblots (25 µg cytosol protein/lane). The cells were probed for FAS and SOX4 mRNA, 18S rRNA, and PR protein as described in the legends of Figs. 6Go and 7Go. The relative levels of FAS and SOX4 (AU) corrected for loading are shown graphically.

 
In cell lines where PRB and PRA were both present, basal levels of SOX4 and FAS mRNA were increased by ORG2058 (N5, B8 cells, Fig. 9Go). The progestin-mediated increase in SOX4 mRNA expression was diminished upon 15- to 20-fold induction of PRA [SOX4 mRNA levels after loading correction: 51% (N5 cells) and 75% (B8 cells) of ORG2058 treated, IPTG untreated, Fig. 9Go]. The progestin-mediated increase in basal FAS mRNA levels was diminished upon induction of PRA with IPTG in N5 and B8 cells [FAS mRNA levels after loading correction: 23% (N5 cells) and 60% (B8 cells) of ORG2058 treated, IPTG untreated, Fig. 9Go].

The effect was greater in N5 cells than B8 cells, and the persistence of PRA upon progestin treatment in N5 cells (described above) may play a role in this effect. The N5 cells were interesting in this regard, and also because in these cells, unlike the other clones, the induction of PRA varied from 3-fold to 15-fold between experiments. In experiments where the induction of PRA in N5 cells was 3-fold, augmentation of progestin-mediated SOX4 mRNA expression was noted, with no consistent effect on FAS mRNA (Figs. 6Go and 7Go). In the experiments described in Fig. 9Go, where 15-fold increases of PRA were noted in N5 cells, inhibition of progestin-mediated induction of both SOX4 and FAS expression was seen. This demonstrates that the effects of PRA induction on progestin-mediated gene expression were related to the cellular content and relative level of PRA, rather than to clonal variation between the cell lines examined.

Taken together, these data showed that when PRA was coexpressed with PRB, in 15- to 20-fold excess, inhibition of progestin-mediated increases in SOX4 and FAS was observed. This was in contrast to the effect of PRA on progestin-mediated increases in SOX4 and FAS mRNA in cell lines where relative expression of PRA was 1- to 3.3-fold that of PRB ( Figs. 6–8GoGoGo).

Repression of Transcription of Transfected Progestin-Responsive Plasmids by PRA in T-47DhPRA Cells
Previous studies have shown that PRA can be a dominant negative inhibitor of PRB activity and also of the activities of other members of the steroid receptor family (14, 15, 18, 20, 21). Those previous observations, in contrast to the present study, were made in transient transfections systems. To determine the effect of PRA induction on exogenously transfected progestin-responsive plasmids in T-47DhPRA cells, reporter gene activity was measured after transfection of pMSG-CAT with or without PRA induction. Treatment of transiently transfected cells with ORG2058 resulted in stimulation of CAT activity in T-47DhPRA clones (14- to 15-fold), comparable with the induction observed in wild-type T-47D cells (Fig. 10Go). Increasing the ratio of PRA:PRB, in favor of PRA (PRA:PRB ratio 2:1, Fig. 10Go, inset), in T-47DhPRA-E3 cells caused little or no change in progestin-induced CAT activity (Fig. 10Go). However, when PRA levels were increased so that the ratio was >4:1, as observed in the T-47DhPRAN5 cells (Fig. 10Go, inset), a 2- to 3-fold decrease in the PR-mediated transcriptional activity was observed (Fig. 10Go). These results suggested that a marked excess of PRA was required to decrease PR-mediated transcriptional activity of an exogenously transfected reporter.



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Figure 10. Repression of Transcription of Transfected Progestin-Responsive Plasmids by PRA in T-47DhPRA Cells

Wild-type T-47D cells and T-47DhPRA cells were transiently transfected with pMSG-CAT and treated with IPTG (5 mM) or vehicle. CAT activity was measured 24 h after treatment with ORG2058 (10 nM) or vehicle. CAT activity was normalized for transfection efficiency using pCH110. Experiments were performed in triplicate and the data expressed as fold induction (± SE). Inset shows PRA/B ratio; +IPTG (stippled bars) and -IPTG (clear bars).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study has documented the generation of new clones of T-47D breast cancer cells, containing predominantly PRB, in which the ratio of PRA:PRB can be altered by induction of PRA. The consequence of PRA induction is an alteration in the ratio of the PR isoforms in favor of PRA, such that the predominant isoform in these cells becomes PRA. Although the mechanisms that control the relative expression of the PR isoforms in vivo are not known, in this study their ratio was maintained over multiple passages (data not shown) and only altered upon induction of PRA by sequestration of the lac repressor by IPTG. The advantages of such cell lines in investigating alterations in the ratio of PRA:PRB are that T-47D cells are well characterized models of PR action, in which a number of endogenous progestin-sensitive endpoints, such as inhibition of cell proliferation and modulation of gene expression, have been described. Stable transfection of a plasmid that can be induced to synthesize PRA allows determination of the magnitude of progestin responses both before and after PRA induction, so that the cellular context remains identical except for the levels of PRA contained. In addition, the ratio of PRA:PRB can be calculated precisely in stable transfectants, unlike transient transfectants where only a proportion of cells contain the introduced plasmid.

Expression of PRA had no effect on cell proliferation in any of the cell lines: cells in which PRA was predominant were equally growth inhibited by progestin as cells in which PRB was the predominant isoform. However, after prolonged progestin exposure, PRA overexpressing cells were more sensitive to the growth-inhibitory effects of progestins and displayed an increase in the maximal growth inhibition observed (data not shown). Whether this was due to a specific effect of PRA or was a consequence of higher total cellular PR concentrations as a consequence of PRA induction is not known. Although there was little effect of altering the ratio of PRA:PRB on cell proliferation, there was a marked effect on cell morphology. Cells in which PRA predominated were rounded upon progestin treatment, phase bright, and loosely adherent to the substratum, from which they detached in large numbers and were detected in the medium. These cells were still viable, as evidenced by their ability to readhere to tissue culture flasks once the progestin had been removed. These changes in morphology suggested that there was a loss of adherent properties of cells in which PRA predominated. Studies in PRA transgenic mice may provide insights into the mechanisms involved: an increase in PRA resulted in disruption of the basal membrane and a decrease in cell-cell adhesion (8). A significant proportion of breast tumors contain levels of PRA markedly higher than those of PRB (7); the findings in this study, that PRA-overexpressing cells had lost adherent properties, suggest that such tumors may demonstrate features of poorer prognosis, and this is borne out by current investigations (our unpublished observations).

The effect was examined of induction of PRA on SOX4 mRNA (22) and FAS mRNA (23), which are endogenous targets of progestin action. Progestin induction of mRNA levels of SOX4, which is directly transcriptionally regulated by progestins (22), was augmented by PRA induction in cells with PRA:PRB ratios which ranged from 1 to 3. Interestingly, PRA alone had no stimulatory effect on SOX4 expression when transfected into PR-negative MCF-7M11 cells. This suggested that the activity of PRA was modulated in the presence of PRB, signaling that the PRA-PRB heterodimer may have a different effect on SOX4 gene expression than that of either homodimer alone and suggesting that the transcriptional effect of progestins on SOX4 gene expression in vivo may be dependent on the cellular ratio of PRA and PRB. Induction of PRA such that PRA:PRB ratios were 1–3 had no consistent effect on the progestin induction of FAS mRNA expression. In some experiments, augmentation of the progestin response was noted, and in others diminution or no effect of the progestin response were observed upon PRA induction. This suggested that the effect of PRA overexpression on progestin inducibility of this transcript, if any, was likely to be modest. Transient transfection of a progestin-regulated reporter also showed that progestin induction of reporter gene activity was unaffected by modest relative increases in PRA. Taken together, these data showed that increases up to 4-fold in the relative PRA levels augmented progestin induction of SOX4 mRNA and had little effect on progestin induction of FAS mRNA expression or progestin stimulation of reporter gene activity, suggesting that the progestin was able to stimulate expression of the endogenous and exogenous targets in this study despite increases of up to 4-fold in the cellular PRA:PRB ratio.

To determine the effect of a large PRA excess on the endpoints examined above, clones were examined that had PRA:PRB ratios of 15–20 (T-47DhPRA-B8 and -N5 cells). Such an excess resulted in inhibition of progestin-mediated effects on SOX4 and FAS mRNA expression, in contrast to the effects seen at lower relative PRA levels. PRA:PRB ratios of greater than 4:1 were associated also with inhibition of progestin- responsive reporter gene activity, as previously shown (14, 15, 18, 20, 27). However, the magnitude of the effect was less than that previously demonstrated and required ratios of at least 4:1 to be manifested. This is in contrast with previous studies using transient transfections, which showed the inhibitory activity of PRA to be elicited at relative levels well below those of PRB, but is more consistent with other evidence that at least equimolar levels of PRA are required to demonstrate significant inhibition of PRB transactivation (10).

The findings of this study, that PRA induction had different effects on different endogenous genes, being ineffective on FAS, yet increasing SOX4 induction, was in keeping with previous conclusions that PRA effects are cell type- and promoter specific. However, there was little evidence that the effects of PRA on endogenous or exogenous targets were dominant, as previously shown. Rather, the data are consistent with the view that PRA can inhibit progestin action, but only when it is present at significant excess over PRB. The previous observations on the powerful negative effect of PRA were made using transient transfections, and this may provide explanations for the discrepancy between this and previous studies. The cell lines used in this study were stably expressing the inducible PRA-encoding plasmid, and consequently the cellular levels of PRA and PRB and hence their ratio, were easily measurable.

Treatment of cells with RU486 revealed the expected antiprogestogenic effects in most contexts on endogenous gene expression. Interestingly, however, an agonist effect on SOX4 expression was noted only after PRA induction. This effect was gene specific, as no agonist effect was noted on FAS expression. This suggested that PRA, when occupied by RU486, may demonstrate transactivation in a gene-specific manner. This is in contrast with previous demonstrations that only antagonist-occupied PRB can activate transcription (16, 18, 28). It must be emphasized that there is no formal evidence in this study that PRA-bound RU486 is mediating the agonist effects on SOX4 expression: an alternative explanation is that induction of SOX4 gene expression may require a minimum level of total PR, which was only reached in this study after induction of PRA. Clarification of the effect on endogenous progestin targets of RU486 when bound to PRA will necessitate use of the MCF-7M11 hPRA cell lines, expressing only PRA, and this forms the focus of future studies.

In summary, this study describes new PR-positive cell lines, in which PRA can be induced so that it becomes the predominant isoform, and the effect of alteration in the PRA:PRB ratio on progestin action. PRA induction had little effect on cell proliferation, but there were striking changes in morphology: cells in which PRA predominated were rounded and displayed reduced adherence to tissue culture flasks, while retaining viability, suggesting that increased PRA expression, as observed in a proportion of breast cancers, may play a role in loss of adhesion observed in malignancy. PRA overexpression was associated with effects on endogenous progestin targets that were dependent on the cellular ratio of PRA:PRB. Increases up to 4-fold in the relative PRA levels augmented progestin induction of SOX4 mRNA and had little effect on progestin induction of FAS mRNA expression, whereas 15- to 20-fold relative PRA levels were associated with diminished progestin responses on both SOX4 and FAS mRNA expression. This suggests that progestin was able to stimulate expression of the endogenous targets in this study despite increases of up to 4-fold in the cellular PRA:PRB levels, whereas extreme alterations in the PRA:PRB ratio, in favor of PRA, were associated with inhibition of progestin action. The results of this study are consistent with the view that PRA expression can fluctuate within a broad range in target cells without influencing the nature of progestin action on downstream targets, but that overexpression of PRA, such as is seen in a proportion of breast cancers, may be associated with inhibition of progestin action and features of poor prognosis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recombinant Plasmids
The pSG5-hPR1 plasmid (2) was a gift of Dr. Pierre Chambon (Strasbourg, France). Plasmids containing the progestin-responsive mouse mammary tumor virus long terminal repeat linked to chloramphenicol acetyltransferase (pMSG-CAT) and bacterial ß-galactosidase sequences, pCH110, were obtained from Pharmacia LKB Biotechnology (North Ryde, Sydney, Australia). The pOP13 hPRA construct was made by excision of the BamHI-BamHI fragment of PR from pSG5-hPR1 and ligation into the BamHI site of pGEM11A(+) (Promega Corp. North Ryde, Australia) creating a NotI site downstream from the PRA coding sequence. A NotI site was created 54 bp upstream from the PRA ATG codon by PCR. The PRA insert was excised from the pGEM11Z(+) vector using the NotI sites and inserted into the NotI sites of the pOP13CAT vector containing lac operon sequences (Lac-Switch inducible mammalian expression system, Stratagene, La Jolla, CA) replacing the CAT sequence. The PRA insert and surrounding regions were sequenced using the ABI 373A, DNA sequencer using ABI Prism dye terminator cycle sequencing ready reaction kit (ABI Advanced Biotechnologies, Inc., Columbia, MD) with AmpliTaq DNA polymerase, FS (Perkin Elmer Corp., Norwalk, CT).

Cell Lines
The ER-positive, PR-positive T-47D cell line (29), (E.G. Mason Research Institute, Worcester, MA) and an ER-positive, PR-negative clone of the MCF-7M cell line (MCF-7M11, a gift from Dr. Anna de Fazio, Garvan Institute of Medical Research, New South Wales, Australia) were cultured in antibiotic-free phenol red-free RPMI 1640 medium or DMEM, respectively, containing 10% FCS and supplemented as described (24). Cells were negative for mycoplasma contamination as determined using the Genprobe rapid detection system (Gen-Probe Inc. San Diego, CA). Cell stocks were passaged regularly to maintain exponential growth.

Construction of Breast Cancer Cell Lines Stably Expressing hPRA
T-47D cells were plated into 162-cm2 peel-back flasks (2.5–3 x 106 cells per flask) in DMEM phenol red-free medium containing 5% FCS 3 days before transfection. The medium was changed on the day of transfection. Cells were transfected using the calcium phosphate precipitation method as described previously (24) with the p3'SS plasmid, encoding the lac repressor (40 µg/162 cm2 flask). T-47D cells were subjected to osmotic shock 3–4 h after transfection, and exposure of T-47D cells to DNA continued for 18 h thereafter (30). Cells were cultured for 6–8 weeks in medium supplemented with hygromycin B (400 µg/ml, Life Technologies, Inc., Melbourne, Australia), and surviving colonies were selected using cloning cylinders. Individual colonies were transferred to 24-well plates and expanded in the presence of hygromycin B (200 µg/ml). Ten clonal cell lines were analyzed for lac repressor protein expression by immunoblot analysis using rabbit anti-LacI polyclonal sera. Three cell lines expressing high, medium, and low amounts of lac repressor protein (T47Dp3'SS -B, -E, and -N, respectively) were transfected with the pOP13 hPRA expression vector (40 µg/162 cm2 flask) and selected using 400 µg/ml geneticin (G418) (Life Technologies, Inc.). T-47D clones containing both the p3'SS vector and the pOP13 hPRA plasmid were maintained in medium supplemented with hygromycin B (200 µg/ml) and G418 (200 µg/ml). MCF-7M11 cells were transfected with the pOP13 hPRA only and selected and maintained with 400 µg/ml and 200 µg/ml G418, respectively.

Cell Growth Experiments
Cells were seeded in 96-well plates (100 µl medium) or 25-cm2 flasks (5 ml medium) in triplicate at a density of 2 x 104 cells/ml. Twenty four hours later (day -1) the medium was supplemented with 10 mM IPTG, to sequester the lac repressor and allow induction of PRA from pOP13 hPRA or vehicle. On day 0, the medium was supplemented with ORG2058 (10 nM) or vehicle only. Cell proliferation was assayed using the CellTiter 96 nonradioactive cell proliferation assay (Promega Corp., Madison, WI) and recorded on days -2, -1, 2, 4, and 6, as the absorbance at 570 nm with a reference wavelength of 630 nm. Cell counts were determined by counting using a hemocytometer on days -2, 0, 3, and 5.

Transient Transfections
T-47DhPRA cells were cotransfected with pMSG-CAT (20–40 µg/150 cm2 flask) and pCH110 (10–20 µg/150 cm2 flask), harvested the next day from the 150-cm2 flasks, and replated into 25-cm2 flasks (8 x 105 cells per flask). Six hours after seeding, cells were treated with 10 mM IPTG or vehicle; 24 h later, cells were treated with the synthetic progestin ORG2058 (10 nM) or vehicle, and enzyme activities were measured subsequently as described previously (30). MCF-7M11PRA cells were transfected with pMSG-CAT (3.5 µg/25 cm2 flask) and pCH110 (1 µg/25 cm2 flask) using Fugene transfection reagent (Boehringer Mannheim, Indianapolis, IN) for 24 h before addition of either ORG2058 (10 nM) or vehicle. All experiments were performed in triplicate.

Immunoblot Analysis of PR
T-47D and T-47DhPRA cells were harvested after treatment with IPTG (Life Technologies, Inc., Melbourne, Australia) 1–10 mM, or vehicle and/or ORG2058, 10 nM and/or RU486 (100 nM) and/or vehicle and stored as described previously (31). Cell pellets were thawed on ice in PEMTG buffer (24) containing 0.4 M KCl and protease inhibitors [0.5 mM phenylmethylsulfonyl fluoride, 1.4 µM pepstatin A, bacitracin (100 µg/ml), 25 mM benzamidine, 86 µM leupeptin, and aprotinin (77 µg/ml)]. Cytosol extracts were prepared and 15–30 µg protein were transferred to nitrocellulose as described previously (Ref. 24 and references therein). Protein concentration was determined by the method of Bradford (Bio-Rad Laboratories, Inc., Reagents Park, Australia). Blots were incubated with monoclonal antibodies against human PR (hPRa3, or 6 and 7) (32) at saturating concentrations and goat antimouse Igs linked to horseradish peroxidase at 1:5000 (Bio-Rad Laboratories, Inc.). PR immunoreactivity was visualized using a chemiluminescent method (ECL, Amersham Pharmacia Biotech, Piscataway, NJ). The relative intensity, in the linear range of the film, of the immunoreactive bands was calculated after densitometric scanning of x-ray films (Molecular Dynamics, Inc., Sunnyvale, CA), followed by integration of the areas under the peaks corresponding to PRA and PRB (Imagequant, Molecular Dynamics, Inc.). Results are expressed as the percentage of immunoreactivity in control samples.

Isolation and Analysis of RNA
Total RNA was extracted using Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH) and transferred to N+ hybond membrane (Amersham Pharmacia Biotech) or Zeta probe (Bio-Rad Laboratories, Inc.) using Vacu-blot (Bio-Rad Laboratories, Inc.) and fixed by UV radiation (Amersham Pharmacia Biotech UV cross-linker). cDNA probes were labeled by random priming using the Amersham Pharmacia Biotech Multiprime DNA labeling system; Northern blots were quantitated using a PhosphorImager and Imagequant software (Molecular Dynamics, Inc.) and subsequently exposed to x-ray film and analyzed as described above. FAS cDNA was a gift from Dr. Henri Rochefort (Montpellier, France). The cDNA probes used for detection were as follows: PR cDNA, containing the full-length hPRA coding sequence and a SOX4 cDNA fragment (nucleotides 1612–2412) (22). The 18S rRNA probe (30-bp oligonucleotide complementary to rat 18S rRNA) (33) and the ß-actin probe from human cDNA plasmid clone, LK221, 2 kb BamHI fragment (a gift from Dr. Linda Bendall (Westmead Institute for Cancer Research, New South Wales, Australia), were used to control for RNA loading.


    ACKNOWLEDGMENTS
 
The authors thank Patricia Mote for assistance with the cell lines, Nham Tran for assistance in preparation of the figures, and James Indsto for assistance with photography.


    FOOTNOTES
 
Address requests for reprints to: Christine L. Clarke, Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia.

Supported by the National Health and Medical Research Council of Australia and the New South Wales Cancer Council. E.M.M. received a Dora Lush Biomedical Research Scholarship from the National Health and Medical Research Council of Australia and an Initiating Grant from the Westmead Hospital Foundation.

Received for publication January 11, 1999. Revision received June 3, 1999. Accepted for publication July 2, 1999.


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