Progestins Inhibit the Growth of MDA-MB-231 Cells Transfected with Progesterone Receptor Complementary DNA1

Vol. 5, 395–403, February 1999 Clinical Cancer Research 395

Valerie C-L. Lin,2 Eng Hen Ng, Swee Eng Aw, INTRODUCTION Michelle G-K. Tan, Esther H-L. Ng, The involvement of progesterone in the growth and devel- Vivian S-W. Chan, and Gay Hui Ho opment of breast cancer has been increasingly recognized in recent years. However, the roles of progesterone in the growth Departments of Clinical Research, Ministry of Health (V. C-L. L., regulation of breast cancer are still controversial. Progestins are S. E. A., M. G-K. T., V. S-W. C.), and General Surgery, Singapore General Hospital (E. H. N., E. H-L. N., G. H. H.), Republic of found to stimulate growth, have no effect, or inhibit growth in Singapore 169608 breast cancer cells (1–13). The conflicting findings affect clinical decision as to whether progestins or antiprogestins would be more appropriate endocrine therapies for PgR3-positive breast ABSTRACT cancer. Although progestins such as MPA and megestrol acetate Because progesterone exerts its effects mainly via estro- are often effectively used in the treatment of breast cancer gen-dependent progesterone receptor (PgR), the expression (14–16), a number of reports advocate that antiprogestins may of progesterone’s effects may be overshadowed by the prim- be new powerful tools for treating hormone-dependent breast ing effect of estrogen. PgR expression vectors were trans- cancer (17–19). fected into estrogen receptor (ER)-␣ and PgR-negative The reasons for the inconsistency in reported effects of breast cancer cells MDA-MB-231; thus the functions of progestins are not clear, but the involvement of estrogen and ER progesterone could be studied independent of estrogens and in the demonstrated effects of progestins is an important factor ERs. Eight stable transfectant clones expressing both PgR to consider. Although PgRs are regulated by a number of hormones and growth factors, they are ER-dependent gene products isoform A and B were studied for their growth response to (20–23). The cells usually need to be primed by estrogenic progesterone and its analogues. Although progesterone had compounds before the effects of progesterone are studied. It is no effect on growth in the control transfectant, the hormone conceivable that the effects of estrogen will be present almost as markedly inhibited DNA synthesis and cell growth in all of -12 -6 long as the effects of progesterone last. Indeed, Otto (24) has the PgR-transfectants dose-dependently from 10 -10 M. shown that a pulse of 1 nM estrogen for 1 min was sufficient to This growth inhibition was associated with an arrest of cells partially stimulate cell proliferation for 5 days. As a result, it is in the G0/G1 phase of the cell cycle. Progestins medroxypro- often difficult to differentiate the specific effects of progester- gesterone acetate, Org2058, and R5020 also strongly inhib- one from that of estrogen, or the expression of progesterone’s ited DNA synthesis, and their doses required for maximal effects may be overridden or masked by the effects of estrogens. -17 -13 -7 inhibition of 60–70% were 10 M,10 M, and 10 M, To delineate the functions of progestins in the regulation of respectively. Antiprogestin ZK98299 alone had no effect, but breast cancer cell growth, we have established ER-independent the compound was capable of counteracting the inhibitory expression of PgR by stably transfecting PgR cDNA into the effect of progesterone. In contrast, RU486 inhibited DNA ER-␣- and PgR-negative breast cancer cell line MDA-MB-231 synthesis, and it showed no further effects when it was used which has recently been reported to express ER-␤ mRNA (25). concurrently with progesterone. These results indicate that The effects of progestins on cell growth and cell cycle kinetics progestins are per se antiproliferative via a PgR-mediated were thus studied in the PgR-positive but ER-negative cell mechanism in breast cancer cells. More importantly, we model. have shown that progestins may exert effective inhibitory Approximately one-third of all breast cancer cases are control over the cell growth if the PgR expression is reacti- ER-negative (26), most of which are also PgR-negative and vated in ER- and PgR-negative breast cancer cells. have aggressive biological behaviors. These patients are insen- sitive to endocrine therapy and are generally associated with poor prognosis. As an initial step toward gene therapy, an important goal of the present study was to determine whether progestin or antiprogestin can exert inhibitory control over growth if the PgR expression is reactivated in ER- and PgR- Received 6/9/98; revised 11/17/98; accepted 11/19/98. negative breast cancer cells. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 These studies were supported by the National Medical Research Coun- cil, Republic of Singapore. 3 The abbreviations used are: PgR, progesterone receptor; MPA, 2 To whom requests for reprints should be addressed , at Department of medroxyprogesterone acetate; ER, estrogen receptor; PgR-B, PgR Clinical Research, Ministry of Health, Singapore General Hospital, isoform B; PgR-A, PgR isoform A; EIA, enzyme immunoassay; Outram Road, Singapore 169608. Phone: 65-3213730; Fax: DCC, dextran-coated charcoal; DCC-FCS, DCC-treated FCS; BrdUrd, 65-2257796; E-mail: [email protected]. bromodeoxyuridine; PI, propidium iodide.

MATERIALS AND METHODS only. The medium containing test compounds was changed Cell Culture. MDA-MB-231 cells and MCF-7 cells every other day, and the cell numbers were determined by were obtained from American Tissue Culture Collection in 1995 counting on a hemocytometer after 5 days’ treatment. 3 at passages 28 and 147, respectively. MDA-MB-231 cells were DNA Synthesis. Cells (3 ϫ 10 ) were seeded onto 96- cloned using 96-well plates by plating 0.5 cell/well. Clone 2 well plates in Test Medium and were treated with test com- (known as MDA-MB-231-CL2) was selected for transfection pounds 2 days later. The effect on DNA synthesis after 48-h studies. All of the cells were routinely maintained in phenol-red treatment was measured by an ELISA kit of BrdUrd incorpora- containing DMEM supplemented with 5% FCS, 2 mM gluta- tion (Boehringer Mannheim) according to the manufacturer’s mine, and 40 mg/l gentamicin. instructions. 5 Chemicals. Progesterone, MPA, and estradiol-17␤ were DNA Flow Cytometry: PI Staining. Cells (1 ϫ 10 )in obtained from Sigma Chemical Co (St. Louis, MO). Org2058 6-well plates were grown in Test Medium for 3 days before they and R5020 were purchased from Amersham (England) and were treated with the test compounds for 24 h. The cells were NEN (Boston, MA), respectively. RU486 (mifepristone) and then harvested and stained with PI in Vindelov’s (28) cocktail ZK98299 (onapristone) were from Siniwest Holdings, Inc. All [10 mM Tris-HCl (pH 8), 10 mM NaCl, 50 mg PI/l, and 10 mg/l of the tissue culture plastics and reagents were obtained from RNaseA, and 0.1% NP40] for 20 min in the dark. The stained Life Technologies, Inc. cells were analyzed in FACS Calibur flow cytometer (Becton Transfection. PgR expression vectors hPR1 and hPR2 Dickinson) with excitation wavelength of 488 nm. The resulting were generous gifts of Professor P. Chambon, Institute of Ge- histograms were analyzed by program MODFIT (Becton Dick- inson) for cell distribution in cell cycle phases. The average netics and Molecular and Cellular Biology, Strasbourg, France. coefficient of variation (CV) is within 5%. Vectors hPR1 and hPR2 contain human PgR cDNA coding for DNA Flow Cytometry: BrdUrd-DNA Analysis. Cells PgR-B and PgR-A, respectively, in pSG5 plasmid (27). Vector (2 ϫ 105) were grown in 60-mm Petri dishes in medium A for pBK-CMV (Stratagene) containing the neomycin-resistant gene 3 days before they received 30 min pulse labeling with 20 ␮M was cotransfected with hPR1 and hPR2 into MDA-MB-231- BrdUrd. The cells were then washed and treated with 1 nM CL2 cells using Lipofectin reagent (Life Technologies, Inc.). progesterone for 6, 16, and 30 h. At the designated time, the Neomycin-resistant clones selected in medium containing G418 cells were harvested by trypsinization, fixed in 70% ice-cold (500 ␮g/ml) were further screened for vector pSG5 sequence by ethanol, and the DNA was denatured into single-stranded mol- PCR using primers flanking the regions of nucleotide 182–405 ecules in 2 N HCl/0.5% Triton X-100. After neutralization with bp, which have little sequence homology to the vector pBK- 0.1 M sodium tetraborate (pH 8.5), the cells were stained with CMV. The PCR product of expected size was further confirmed FITC-labeled anti-BrdUrd (Becton Dickinson) for 30 min at by digestion with restriction enzyme NcoI. Levels of PgR in room temperature in PBS containing 0.5%Tween-20/1% BSA. pSG5-positive transfectants were determined using the PgR EIA After washing with PBS/0.5%Tween-20/1% BSA, the cells kit from Abbott Laboratories. Cells stably transfected with both were stained with 5 ␮g/ml of PI and analyzed on FACS Calibur vector pBK-CMV and pSG5 plasmid were used as transfection flow cytometer for incorporated BrdUrd (FITC) and total DNA controls. content (PI) simultaneously. Western Blotting Analysis. Total proteins were ex- Statistical Analysis. Differences between treatments tracted from the transfected cells by three cycles of freezing were tested by ANOVA. When significant differences were (liquid nitrogen) and thawing (37°C water bath) in buffer con- detected by ANOVA, multiple comparisons among means were taining 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM DTT, and performed by the least significant difference test. Correlation a cocktail of protease inhibitors for serine, cysteine, and metal- analysis was performed using the program in Microsoft Excel. loproteases (Boehringer Mannheim, Mannheim, Germany). Cy- tosols containing 50 ␮g of protein were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes. After RESULTS blocking overnight in Tris-buffered saline containing 0.05% Characterization of Transfectants. ER-␣- and PgR- Tween-20 and 3% BSA, the membrane was probed using PgR negative breast cancer cells MDA-MB-231-CL2 were trans- antibodies from Abbott Laboratories. The specific bands corre- fected with PgR expression vectors hPR1 and hPR2. Vector

sponding to PgR-A (Mr 90,000) and PgR-B (Mr 120,000) were pBK-CMV was cotransfected with PgR expression vectors be- detected with enhanced chemiluminescence (ECL) kit (Amer- cause it contains neomycin-resistant gene as selection marker sham). for initial screening. Eight transfectant clones expressing both ϫ 4 Cell Growth. Cells (2 10 ) in their late exponential PgR-A (Mr 90,000) and PgR-B (Mr 120,000) were generated as growth phase were seeded onto the 12-well plates in phenol shown by Western blotting analysis (Fig. 1). The identity of the

red-free DMEM supplemented with 2 mML-glutamine, 40 mg/l Mr 80,000 band is not clear. Because the band is absent in gentamicin, and 5% (DCC-FCS; Test Medium). The treatment PgR-negative control transfectant CTC15 cells, it is possible of FCS with DCC was to remove from FCS the endogenous that it represents the proteolytic fragments of PgR. The ratios of steroid hormones that may complicate the effects of progestins the two isoforms expressed differ among the transfectant clones. and antiprogestins. Two days later, the medium was replaced The transfectant clones ABC3, ABC4, ABC5, ABC13, ABC21, with fresh medium containing test compounds that were added ABC24, ABC28, and ABC79 expressed 456, 815, 149, 517, from 1000-fold stock in ethanol. This gave a final concentration 1429, 1992, 657, and 488 fmol/mg protein, respectively, as of ethanol of 0.1%. Treatment controls received 0.1% ethanol analyzed by EIA. By comparison, the levels of PgR reported for

Fig. 1 Western blotting analysis of total proteins extracted from both PgR-A- and PgR-B-transfected clones and control transfectant CTC15 cells. Cytosols containing 50 ␮g of protein were separated in 7.5% SDS-polyacrylamide gel and transferred onto nylon membrane. Molec- ular weight markers are indicated on the left. PgR was detected using the antibody from PgR EIA kit of Abbott Laboratories. The specific bands corresponding to A isoform (Mr 90,000)and B isoform (Mr 120,000) of PgR are indicated by arrows. The identity of the band at approximately Mr 80,000 is unknown. Lanes ABC3 –ABC79 are the eight PgR- transfected clones and CTC15 is the vector-transfected control in which no PgR was detected. Arrows in Lanes ABC13 and ABC21 are nonspe- cific signals from the X-ray film.

T47-D cells were 1000–3000 fmol/mg protein, and for estradiol-treated MCF-7 cells were 136–326 fmol/mg protein (1, 7, 9). Clone CTC15 transfected with both pSG5 and pBK-CMV plas- mids was used as transfectant control. No PgR were detected in CTC15 by either EIA or Western blotting analysis. Effect of Progesterone on the Growth of PgR-transfected Cells. We focused our study on PgR-transfectant clone ABC28 initially because it expressed a moderate number of PgRs. The effects of progesterone on other PgR-transfected clones will be reported in the latter part of the text. Fig. 2 illustrates the effect of various concentrations of progesterone on BrdUrd incorporation (BrdUrd incorporation will also be referred to as DNA synthesis in the rest of the article) and cell growth of PgR–transfected clone ABC28 cells and the controls. The results are expressed as percentage of vehicle–treated controls. Measured 2 days after treatment, progesterone signifi- Fig. 2 The effects of various concentrations of progesterone on cantly (P Ͻ 0.01) inhibited BrdUrd incorporation of ABC28 DNA synthesis and on cell growth of PgR-transfected clone ABC28, -11 vector-transfected control CTC15 cells, and the parent cell line cells from 10 M, and the inhibitory effect increased with MDA-MB-231-CL2 cells. In a, DNA synthesis was measured by increasing concentrations of the hormone (Fig. 2a). Maximal ELISA of BrdUrd incorporation. Cells were plated onto 96-well -5 inhibition (80%) of DNA synthesis was achieved with 10 M. plates (3000 cells/well) in phenol red-free DMEM supplemented Similarly, progesterone also reduced the growth of the PgR with 5% DCC-FCS. Progesterone containing medium was added to the cells 2 days later at various concentrations in 0.1% ethanol. transfectants measured by a cell-counting experiment in a con- BrdUrd incorporation was measured 48 h later by ELISA according centration-dependent fashion (Fig. 2b). Maximal inhibition of to manufacturer’s instructions. In b, cell growth was determined by -9 cell growth was observed with 10 M, and the cell number was cell counting. Cells (2 ϫ 104) were plated onto 12-well plates in 1 ml reduced by 70% after 5 days’ treatment. In contrast, progester- of phenol red-free DMEM supplemented with 5% DCC-FCS. Pro- one had no significant effect on cell growth in transfectant gesterone-containing medium was added to the cells 2 days later at various concentrations in 0.1% ethanol. The medium containing control clone CTC15 cells and the parental cell line MDA-MB- progesterone was changed every 2 days. The cells were harvested -12 -6 231-CL2 at concentrations between 10 M and 10 M. It should after 5 days of treatment, and cell numbers were determined by -5 be noted, however, that progesterone at 10 M increased the cell counting on a hemocytometer. The results are expressed as percent- Ϯ ϭ number significantly in both controls. The mechanism for this ages of vehicle-treated controls (mean SE, n 4). c, the effect of progesterone on the growth of PgR-transfected clone ABC28 during increase is not clear. The growth inhibitory effect of progester- 9 days of culture. Cells were plated in 24-well plates at 10,000/well one in the transfectant is further illustrated in Fig. 2c, which in 0.5 ml of phenol red-free DMEM supplemented with 5% DCC- shows that progesterone-treated ABC28 cells only doubled its FCS. Progesterone (P)-containing medium was added to the cells 2 population over 9 days as compared with over 6-fold increase in days later at concentrations of 0, 0.1 nM, and 0.1 ␮M in 0.1% ethanol. vehicle-treated controls. The medium containing progesterone was changed every 2 days. The cell numbers were determined by counting on a hemocytometer at 0, The growth inhibitory effect of progesterone was also 2, 4, 7, and 9 days after treatment. The results are expressed as observed in seven other PgR-transfectants as shown in Fig. 3. mean Ϯ SE, n ϭ 4.

Fig. 4 Progesterone-induced changes in the cell cycle distributions of PgR-transfected clone ABC28. Cells (1 ϫ 105) in 6-well plates were Fig. 3 The effect of progesterone on DNA synthesis (a) and cell grown in 0.5 ml of phenol red-free DMEM supplemented with 5% growth (b) in eight clones of MDA-MB-231-CL2 cells transfected with DCC-FCS for 3 days before they were treated with progesterone at PgR cDNA and in control transfectant CTC15. DNA synthesis was various concentrations for 24 h. The cells were then harvested and measured by ELISA of BrdUrd incorporation and cell growth was stained with PI in Vindelov’s cocktail [10 mM Tris-HCl (pH 8), 10 mM determined by cell counting on a hemocytometer. Methods are as NaCl, 50 mg PI/l, and 10 mg/l RNaseA, and 0.1% NP40]. The stained described in Fig. 2. Doses of progesterone tested are 1 nM and 1 ␮M. cells were analyzed in FACS Calibur flow cytometer (Becton Dickin- The results are expressed as percentage of vehicle–treated controls Ϯ son) with excitation wavelength of 488 nm. The resulting histograms SE, n ϭ 4. were analyzed by program MODFIT (Becton Dickinson) for cell distribution in cell cycle phases. The average coefficient of variation (CV) is within 5%. Results are expressed as mean Ϯ SE, n ϭ 4.

-9 -6 Progesterone at 10 M and 10 M significantly (P Ͻ 0.01) inhibited DNA synthesis (Fig. 3a) and cell growth (Fig. 3b)in all of the PgR-transfectants. The inhibition varied from about days (Fig. 6). The reduction of S phase cells was paralleled by

15% to 75% depending on the clones tested. More interestingly, the accumulation of cells in G0-G1 phase. The proportion of Ͻ significant (P 0.05) correlation was observed between the cells in G2-M phase in progesterone-treated cells began to extent of growth inhibition by progesterone and PgR concen- decrease after 36 h of treatment. This decrease in G2-M phase trations analyzed by EIA. cells is likely the consequence of the accumulation of G0-G1 Effect of Progesterone on Cell Cycle Kinetics. The phase cells. growth-inhibitory effect of progesterone in PgR-transfected The reductions of S phase cells in progesterone-treated MDA-MB-231-CL2 cells was associated with dose-dependent ABC28 cells were also demonstrated in low serum condition reductions of the percentage of S phase cells, accompanied by under which the cells were slowly proliferating. As is shown in

an increase in G0-G1 phase cells (Fig. 4). To investigate in more Table 1, the S phase fractions in progesterone-treated ABC28 detail the time course of changes in the distribution of cell cycle cells grown in phenol red-free medium containing 1% DCC- phases, ABC28 cells were analyzed for the distribution of cell FCS were reduced to approximately 12.5% after 15 h and 18 h cycle phases at 3–6-h intervals for a 48-h period after proges- of treatment, and to 10% after 24 h and 48 h of treatment. This terone treatment (Fig. 5). As expected, vehicle-treated cultures is similar to the S phase fractions of progesterone-treated ABC displayed cyclic changes in distribution of cell cycle phases, 28 cells in medium containing 5% DCC-FCS, but the S phase most obviously seen in the percentage of S phase cells. Progres- fractions in the vehicle-treated control cells were approximately sive reduction of S phase cells began at 12 h after treatment. 20–30% lower in medium containing 1% serum as compared Maximal reduction (65%) of S phase cells was achieved by 36 h. with the cells in medium containing 5% serum.

The S phase cells in progesterone-treated cultures was main- The arrest in G0-G1 phase is further illustrated by the tained at 40–50% of the vehicle-treated control during the 6-day analysis of BrdUrd/DNA distributions measured for ABC28 period, during which time the medium was changed every 2 cells at several time points after progesterone treatment (Fig. 7).

Fig. 5 Time course of progesterone-induced changes in the distribution of Fig. 6 Progesterone-induced changes in cell cycle distribution of PgR- cell cycle phases in PgR-transfected clone ABC28. Cells (1 ϫ 105)in transfected clone ABC28 cells during 6 days in culture. Cells (1 ϫ 105) 6-well plates were grown in phenol red-free DMEM supplemented with 5% in 6-well plates were grown in phenol red-free DMEM supplemented DCC-FCS for 3 days before they were treated with 0.1% ethanol (E)or1 with 5% DCC-FCS for 3 days before they were treated with 0.1% nM progesterone in 0.1% ethanol (F). Cells were collected for cell cycle ethanol (E)or1nM progesterone in 0.1% ethanol (F). Cells were analysis at various times after the addition of progesterone. Methods are as collected for cell cycle analysis after 0, 2, 4, and 6 days of treatment with described in Fig. 5. Results are expressed as mean Ϯ SE, n ϭ 4. progesterone. Methods are as described in Fig. 5. Results are expressed as mean Ϯ SE, n ϭ 4.

Vehicle-treated controls are displayed in parallel with progest- of the PgR-transfectants responded to progesterone with signif- erone-treated samples at each time point. The cells were labeled icant reductions in the percentage of S phase cells and accom- ␮ with 20 M BrdUrd for 30 min before treatment. Two popula- panying increases in G0-G1 phase cells. In contrast, no effect of tions of G0-G1 cells are seen at 16 h after treatment; the top progesterone was seen in cell cycle distribution of the control portion are BrdUrd-labeled cells that were in S phase at the time transfectant CTC15 cells and the parent cell line MDA-MB- of BrdUrd pulse; the population at the lower portion are unla- 231-CL2.

beled cells that were in G0-G1 or G2-M phase at the time of Effect of Various Progestins, Antiprogestins, and Estra- BrdUrd addition. It can be seen that many fewer unlabeled cells diol on DNA Synthesis of PgR-transfectant. Our study with progressed into S phase in progesterone-treated cells than in eight clones of PgR-transfected cells has shown that the inhib- controls at 16 h after treatment. On the other hand, at this time itory effect of progesterone on BrdUrd incorporation correlated Ͻ (16 h) BrdUrd-labeled cells could progress to G2-M phase and well (P 0.001) with that of cell number determination. subsequently into G0-G1 phase normally in progesterone-treated BrdUrd incorporation was, therefore, used to measure the effect sample as compared with the controls. At 30 h after treatment, on the growth of various synthetic progestins and antiprogestins the cells had completed one cycle after the addition of proges- in PgR-transfectant clone ABC28. Progestins MPA, Org2058, terone and the second cycle began as indicated by the labeled and R5020 inhibited DNA synthesis of PgR–transfectant cells progressing into S phase. There were considerably fewer ABC28 cells, and the inhibition is concentration-dependent number of cells progressing to S phase in progesterone-treated (Fig. 9a). The potencies were in the order: MPA Ͼ Org2058 Ͼ cells when compared with the controls at 30 h after treatment in progesterone Ͼ R5020. MPA was by far the most potent growth -17 both the BrdUrd-labeled and -unlabeled populations. Conse- inhibitor; the inhibitory activity begins with 10 M, and this is

quently, there was also a reduction of G2-M phase cells in 100,000 times more potent than the natural compound proges- progesterone-treated cells. terone. Of special mention is that fact that all of the four Fig. 8 illustrates progesterone-induced cell cycle changes progestins inhibit BrdUrd incorporation to a maximum of 70% in five other PgR-transfectant clones and the controls after the of the vehicle-treated controls, although their effective doses -12 -9 -6 24 h treatment at different doses (0, 10 M,10 M,10 M). All may vary by several thousand fold.

Table 1 Progesterone-induced changes in S phase fraction of PgR-transfected clone ABC28 cells in low-serum medium Cells (1.2 ϫ 105) in 12-well plates were grown in phenol red-free DMEM supplemented with 1% DCC-FCS for 24 h before they were treated with vehicle only (0.1% ethanol) or with 1 nM progesterone. Cells were collected for cell cycle analysis using flow cytometer after 15 h, 18 h, 24 h, and 48 h of treatment. Methods are as described in Fig. 5. Results are expressed as mean Ϯ SE, n ϭ 4. Time (h) Treatment 15 18 24 48 Vehicle-treated cells 19.6 Ϯ 0.17 21.9 Ϯ 0.33 26.1 Ϯ 0.31 20.9 Ϯ 0.54 Progesterone-treated cells 12.2 Ϯ 0.48 12.4 Ϯ 0.07 10.6 Ϯ 0.33 10.9 Ϯ 0.25

Fig. 7 Flow cytometry analysis of BrdUrd-DNA distribution of clone ABC28 cells at 6, 16, and 30 h after progesterone treatment. Cells (2 ϫ 105) were grown in 60-mm Petri dishes in phenol red-free medium Fig. 8 Effects of various concentrations of progesterone on the distri- supplemented with 5% DCC-FCS for 2 days before they were treated for bution of cell cycle phases in different PgR-transfected clones and the 5 30 min with 20 ␮M BrdUrd. The cells were then washed and treated with control cells. Cells (1 ϫ 10 ) in 6-well plates were grown in phenol 1nM progesterone. At the designated time, the cells were harvested and red-free DMEM supplemented with 5% DCC-FCS for 3 days before stained with FITC-labeled anti-BrdUrd antibody and PI (see “Materials they received different concentrations of progesterone treatments as are and Methods” for details), and analyzed on FACS Calibur flow cytom- depicted in the graph. Cells were collected for cell cycle analysis after eter for incorporated BrdUrd (FITC) and total DNA content (PI) simul- 24 h of treatment. Methods are as described in Fig. 5. Results are taneously. expressed as percentage of vehicle–treated controls Ϯ SE, n ϭ 4.

On the other hand, antiprogestins RU486 and ZK98299 showed different effects on the growth of PgR-transfectants. effect of progesterone on ABC28 cells (Fig. 9a). There appeared ␤ ZK98299 alone was without any effect over the concentration to be, therefore, no ER- -mediated events in PgR-mediated -12 -6 range of 10 –10 M, but the compound was capable of coun- growth inhibition of ABC28 cells. teracting the inhibitory effect of progesterone as illustrated in Fig. 9b. In contrast, RU486 inhibited DNA synthesis from DISCUSSION -12 -6 10 –10 M, and it had no further effects when it was concur- We have stably transfected PgR cDNA into ER and PgR- rently used with progesterone. Estradiol-17␤ at concentrations negative MDA-MB-231 cells to delineate the growth effect of -12 -6 of 10 –10 M showed no effect on DNA synthesis in ABC28 progesterone in the absence of estrogen and/or ER. These PgR cells. transfectants expressed both isoforms A and B receptors with It is also interesting to note that estradiol-17␤ at concen- varying A:B ratios. Our study has convincingly demonstrated -11 -6 trations of 10 –10 M did not modify the growth inhibitory that progesterone and other synthetic progestins markedly in-

provided evidence that some progestins (progesterone, Org2058, Org 30659, gestodene, levonorgestrel, 3-ketodeso- gestrel, and norethisterone) stimulate the growth of breast cancer cells by ER-mediated mechanisms (5, 6, 10, 13, 29). Our goal was to establish a PgR-positive but ER-negative cell model that would enable us to isolate the growth effects of progesterone from that of estrogens, and to differentiate between PgR-mediated effects and ER-mediated effects. The results using the PgR-transfected MDA-MB-231 cells have clearly showed that progestins inhibit the growth of breast cancer cells via PgRs that are expressed independent of ER and estrogens, thus supporting the notion that the growth stimulatory effect could be ER-mediated. Although PgR concentrations in PgR-transfected MDA- MB-231 cells are similar to what was reported for T-47D cells -17 -14 -12 -9 (1, 7, 9), the effective doses (10 M,10 M,10 M, and 10 M for MPA, Org2058, progesterone and R5020, respectively) for the inhibition of growth of PgR-transfected cells are much lower than what was reported for T47D cells (1, 7). This may be due to the absence of the counteracting effect of estrogens or the absence of ER-mediated effects. The molecular basis for the varying potency of these progestins is not clear, and we have not performed ligand-binding studies to determine the binding af- finity of each of these compounds to PgR. Flow cytometry analysis revealed that progesterone- induced cell cycle changes in PgR-transfected MDA-MB-231 cells were characterized by significant reduction of S phase fraction and an accumulation in G -G phase beginning at 12 h Fig. 9 a, the effects of various synthetic progestins, antiprogestins and 0 1 estradiol-17 ␤ on DNA synthesis of PgR-transfected clone ABC28. The after the treatment with progesterone. This response is similar to DNA synthesis was determined by ELISA of BrdUrd incorporation. that reported by Sutherland et al. (1) for T-47D cells induced by Cells (3000) were plated onto 96-well plates in phenol red-free DMEM MPA, which exhibited growth inhibitory effect. However, under supplemented with 5% DCC-FCS. Test compounds-containing medium experimental conditions in which slowly proliferating cells are was added to the cells 2 days later at various concentrations in 0.1% tested, progestins caused a biphasic change in the cell cycle ethanol. BrdUrd incorporation was measured 48 h later by ELISA according to manufacturer’s instructions. Results are the means (n ϭ 4) progression of T47-D cells: the cell growth was accelerated of percentage of vehicle-treated controls. The average SE is 6.7%. b, through the first cell cycle but arrested in late G1 phase of the effects of antiprogestins RU486 and ZK98299 on progesterone-induced second cycle (8, 30). Groshong et al. (31) showed similar inhibition of DNA synthesis in PgR-transfectant clone ABC28. Methods biphasic regulation by progesterone of the growth in T-47D-YA are as described in a. Results are presented for treatment with progesterone, RU486, or ZK98299 alone at various concentrations, or for or T-47D-YB cells, which constitutively express the B or A treatment with 1 nM progesterone (P) in the presence of various con- isoform of PgR (32). These are in contrast with our findings that centrations of RU486 or ZK98299. Results are the means (n ϭ 4) of progesterone consistently inhibited the growth of PgR-trans- percentage of vehicle-treated controls. The average SE of the means is fected MDA-MB-231 cells when the cells were tested under low 7.5%. serum (1%) condition after 15 h, 18 h, 24 h, and 48 h of treatment. It is interesting to note that antiprogestin ZK98299 re- hibited DNA synthesis and cell growth in these PgR transfec- versed the growth inhibitory effect of progesterone in PgR- tants. The growth inhibition is specifically PgR-mediated since transfected MDA-MB-231 cells whereas antiprogestin RU486 the control transfectant and the parent cell line MDA-MB-231 did not. Instead, RU486 exhibited agonist activity to inhibit the cells showed no specific response to progesterone treatment. growth. This finding is in accord with the theory that ZK98299 Furthermore, the growth inhibitory effect of progesterone was and RU486 belong to two different mechanistic types of anti- PgR concentration-dependent because the magnitude of growth progestin (33, 34). Type II antiprogestin, represented by RU486, inhibition in eight PgR-transfected clones positively correlated can switch its property from an antagonist to an agonist under with PgR concentrations. the influence of a certain signaling network (32, 35) and the One unique feature in the classical model for the mech- steroid receptor interacting proteins (36). anism of the action of progesterone is that the hormone acts It is known that ovarian steroid hormones play an im- via estrogen-dependent PgR (20–23). According to this portant role in the growth and development of breast cancer. model, progesterone can exert an effect only in the cell that Antiestrogen tamoxifen is presently the front line therapy for has already been stimulated by estrogens. As a result, the ER- and PgR-positive breast cancer (37, 38). On the other effect of progesterone can often be masked or overridden by hand, one-third of all breast cancer cases are presented with the effect of estrogen. Furthermore, a number of studies ER- and PgR-negative disease (26). One potential therapeutic

approach for this group of patients is to introduce exogenous 11. Dauvois, S., Simard, J., Dumont, M., Haagensen, D. E., and Labrie, ER or PgR genes to breast cancer cells so that hormonal F. Opposite effects of estrogen and the progestin R5020 on cell prolif- control can be reestablished (39, 40, 41). Jiang and Jordan eration and GCDFP-15 expression in ZR-75–1 human breast cancer (42) have shown that estrogens can inhibit the growth of ER cells. Mol. Cell. Endocrinol., 73: 171–178, 1990. cDNA-transfected MDA-MB-231 cells. Our study also indi- 12. Hissom, J. R., and Moore, M. R. Progestin effects on growth in the human breast cancer cell line T-47D:possible therapeutic implications. cates that progestins are able to exert strong inhibitory con- Biochem. Biophys. Res. Commun., 145: 706–711, 1987. trol over the growth in vitro if the PgR expression is restored 13. Cappeletti, V., Miodini, P., Fioravanti, L., and Difronzo, G. Effect in ER- and PgR-negative breast cancer cells. 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