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Westmead Institute for Cancer Research University of Sydney Westmead Hospital Westmead, New South Wales 2145, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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. 1
; 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. 6–8 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. 1) 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. 1): 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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) 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. 2) 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. 2C, inset). There was no
difference in PRA induction at the IPTG concentrations tested (Fig. 2B), 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. 2, 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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) and to that of wild-type T-47D cells
(data not shown). Induction of PRA had no effect on cell proliferation
(Fig. 3, 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. 3A, 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. 3B, 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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, D and F) compared with cells treated
with progestin without PRA induction (Fig. 4, C and E). Cells that
detached from the monolayer retained viability, as evidenced by the
assay shown in Fig. 3A
, 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. 4, compare A and B).
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). Progestin
treatment of MCF-7M11 hPRA cells resulted in down-regulation of ER mRNA
expression (Fig. 5A), a previously described effect of progestin on ER
expression (25), and increased activity of the transiently transfected
pMSG-CAT plasmid (Fig. 5B), 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. 5).
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) and FAS (Fig. 7) mRNA was measured. Addition of IPTG
increased PRA levels in the T-47DhPRA cells but not in the T-47DN
parent cell line (Fig. 6A). 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. 6A, 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. 6A, 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. 6), and -N4
(data not shown) cell lines. Induction of PRA augmented the induction
of SOX4 mRNA expression in T-47DhPRAE3 and -N5 (Fig. 6, A and B) and
-N4 cells (data not shown). There was no effect of PRA induction on the
parent N cells (Fig. 6, 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. 6B). The relationship between
PRA:PRB ratio and SOX4 mRNA expression was explored in a number of
separate experiments (Fig. 6C): 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. 7). Over a range of experiments,
where the same RNA shown in Fig. 6C was probed for FAS, increased PRA
levels had a variable effect on ORG2058 induction of FAS (Fig. 7B),
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. 7C).
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. 8. 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. 6 and 7. RU486 alone had little
effect on FAS mRNA levels, with or without PRA induction (Fig. 8),
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. 8, 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. 8, 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. 8, 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. 6–8 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. 9).
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). 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. 9]. 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. 9].
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. 6 and 7). In the experiments
described in Fig. 9, 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. 6–8).
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. 10). Increasing the ratio of PRA:PRB,
in favor of PRA (PRA:PRB ratio 2:1, Fig. 10, inset), in
T-47DhPRA-E3 cells caused little or no change in progestin-induced CAT
activity (Fig. 10). However, when PRA levels were increased so that the
ratio was >4:1, as observed in the T-47DhPRAN5 cells (Fig. 10, inset), a 2- to 3-fold decrease in the PR-mediated
transcriptional activity was observed (Fig. 10). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, Vehicle; 
, 1
mM IPTG; 
, 5 mM IPTG; 
, 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.
