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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1569
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 953-961
Copyright © 2005 by The Endocrine Society

Progesterone Receptor Modulator CDB-2914 Down-Regulates Proliferative Cell Nuclear Antigen and Bcl-2 Protein Expression and Up-Regulates Caspase-3 and Poly(Adenosine 5'-Diphosphate-ribose) Polymerase Expression in Cultured Human Uterine Leiomyoma Cells

Qin Xu, Shigeki Takekida, Noriyuki Ohara, Wei Chen, Regine Sitruk-Ware, Elof D. B. Johansson and Takeshi Maruo

Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; and Center for Biomedical Research, The Population Council, New York, New York 10021

Address all correspondence and requests for reprints to: Dr. Takeshi Maruo, Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan. E-mail: maruo{at}kobe-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was conducted to evaluate the effects of the progesterone receptor modulator CDB-2914 on proliferative activity and apoptosis in cultured human uterine leiomyoma cells. Isolated leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for 12, 24, 48, and 96 h in the absence or presence of graded concentrations of CDB-2914 (10–9, 10–8, 10–7, and 10–6 M). The number of viable cultured leiomyoma cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide assay. Proliferating cell nuclear antigen (PCNA) expression was evaluated by immunocytochemistry and Western blot analysis. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay. Caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), and Bcl-2 expression were assessed by Western blot analysis. Compared with untreated control cultures, treatment with CDB-2914 decreased the number of viable cultured leiomyoma cells and the PCNA-positive rate in those cells and increased the TUNEL-positive rate in cultured leiomyoma cells in a dose-dependent manner. Western blot analysis revealed that treatment with CDB-2914 significantly decreased the expression of PCNA and Bcl-2 protein and increased the expression of cleaved caspase-3 and cleaved PARP in a dose-dependent manner compared with untreated control cultures. These results suggest that CDB-2914 inhibits the proliferation of cultured leiomyoma cells by down-regulating PCNA expression and induces apoptosis by up-regulating cleaved caspase-3 and PARP expression and down-regulating Bcl-2 protein expression in those cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UTERINE LEIOMYOMA IS a benign smooth muscle tumor originated from myometrium. Leiomyoma has been thought to be an estrogen-dependent tumor because of its frequent occurrence during reproductive age and its regression after menopause. However, several lines of evidence have implicated a critical role for progesterone (P4) in leiomyoma growth (1, 2, 3, 4, 5). The effects of P4 on target tissues are mediated by P4 receptor (PR), which belongs to the nuclear receptor family. PR functions as a ligand-activated transcription factor to regulate the expression of specific sets of target genes (6). PR modulators regress the biological actions of P4 by inhibiting PR activation (6). In this context, RU486 (mifepristone) has been shown to cause a significant regression of the size of leiomyomas (7, 8, 9, 10, 11), but undesirable side-effects related to the antiglucocorticoid effect of RU486 are reported to occur during the treatment, as evidenced by the rise in serum testosterone, androstenedione, and dehydroepiandrosterone sulfate (8, 11). This antiglucocorticoid effect of RU486 is considered neither necessary nor even useful in the long-term treatment of leiomyoma (10).

CDB-2914 (17{alpha}-acetoxy-11ß-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione) is a novel PR modulator that binds competitively to PR with high affinity (12) and has little or no antiglucocorticoid activity (12). The lesser antiglucocorticoid activity of CDB-2914 compared with that of RU486 (12, 13, 14, 15) may provide considerable advantage for the long-term treatment of leiomyoma.

The molecular mechanism by which PR modulators inhibit leiomyoma growth remains to be elucidated. However, recent studies have demonstrated that antiprogestins can inhibit the growth of leiomyoma cells (16) and induce apoptosis of endometrial cells (17) and granulosa cells (18, 19, 20). The apoptotic cell death program is initiated and activated as a result of diverse internal and external signals. These signals include two pathways: a mitochondria-mediated pathway and a ligand-mediated death pathway (21). Upon triggering of either pathway, caspases, which are the final executioners of apoptosis, are activated and cause degradation of cellular proteins and disassembly of cells (22). Caspase-3 is responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP) (23), during the course of apoptosis. PARP is implicated in DNA replication, transcription, DNA repair, apoptosis, and genome stability (24). During apoptosis, caspase-3 causes the cleavage and inactivation of PARP (25, 26, 27). The B cell leukemia/lymphoma-2 (bcl-2) protooncogene was first identified in lymphoma tumors composed of B cells (28). Bcl-2 resides in the outer mitochondrial membrane, the membrane of endoplasmic reticulum, and its associated nuclear envelop (29) and acts to prevent the release of apoptogenic proteins from mitochondria (30). Our recent studies have demonstrated that Bcl-2 protein expression in cultured leiomyoma cells is remarkably up-regulated by P4 (1), whereas TNF{alpha} expression in those cells is down-regulated by P4 (3). These results suggest that P4 may act as an apoptosis-inhibiting factor in leiomyoma cells through cross-talk with Bcl-2 protein and TNF{alpha} in those cells.

In the present study we have demonstrated that CDB-2914 inhibits the proliferation of cultured leiomyoma cells by down-regulating proliferating cell nuclear antigen (PCNA) expression and induces apoptosis by up-regulating cleaved caspase-3 and cleaved PARP expression and down-regulating Bcl-2 protein expression in those cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection

Twenty-three uterine leiomyoma tissues were obtained from women with regular menstrual cycles who underwent hysterectomy or myomectomy for uterine leiomyomas at Kobe University Hospital. Informed consent was obtained from each patient before surgery for the use of uterine tissues for the present study. The institutional review board approved the use of uterine tissues for culture experiments. The patients ranged in age from 35–42 yr, with a mean age of 37.4 yr, and had received no hormonal therapy for at least 6 months before surgery. The histological diagnosis of each uterine specimen was examined. Samples were excluded from the study if accurate menstrual cycle dates could not be assigned or if unexpected pathology was found (e.g. adenomyosis). Thirteen samples were collected from the proliferative phase of the menstrual cycle, and 10 samples were obtained from the secretory phase of the menstrual cycle.

Cell culture

Uterine leiomyoma tissues, dissected from endometrial cell layers, were cut into small pieces and digested in 0.2% collagenase (wt/vol) at 37 C for 3–5 h, as previously described (1). Leiomyoma cells were collected by centrifugation at 460 x g for 5 min and washed three times with PBS containing 1% antibiotic solution. Cell viability was determined by trypan blue exclusion test. The isolated leiomyoma cells were plated at densities of approximately 1 x 106 cells/dish in 10-cm2 culture dishes, 4 x 104 cells/well in two-well chamber glass slides, and 1 x 104/well in 96-well tissue culture plates. The isolated leiomyoma cells in culture dishes and two-well chamber slides were subcultured at 37 C for 120 h in a humidified atmosphere of 5% CO2-95% air in phenol red-free DMEM supplemented with 10% fetal bovine serum (vol/vol; Invitrogen Life Technologies, Inc., Grand Island, NY). The isolated leiomyoma cells in 96-well tissue culture plates were subcultured for 72 h under the conditions described above. The monolayer cultures at approximately 70% confluence were treated with graded concentrations (10–9, 10–8, 10–7, and 10–6 M) of CDB-2914 (Laboratoire HRA Pharma, Paris, France) in serum-free, phenol red-free DMEM for 12, 24, 48, and 96 h. CDB-2914 was dissolved in absolute ethanol.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide (MTT) assay for cell growth and viability

The number of viable leiomyoma cells was evaluated by MTT assay. Briefly, after being treated in the presence or absence of graded concentrations of CDB-2914 (10–9, 10–8, 10–7, and 10–6 M) in serum-free DMEM for 48 and 96 h in a 96-well tissue culture plate, 10 µl MTT (Chemicon International, Inc., Temecula, CA) solution was added to each well, and cultured cells were incubated at 37 C for another 4 h. Then 100 µl isopropanol/HCl solution were added to each well and mixed thoroughly with a microtube mixer. Absorbance was measured in an MTP-120 ELISA plate reader (Corona Electric Co., Osaka, Japan) with a test wavelength of 570 nm and a reference wavelength of 630 nm.

Immunocytochemical staining for PCNA

Leiomyoma cells cultured in two-well chamber slides were washed three times with PBS, fixed in methanol at 4 C for 20 min, and again washed with PBS three times. The fixed cells were subjected to immunostaining by the avidin/biotin immunoperoxidase method using a polyvalent immunoperoxidase kit (Omnitags, Lipshow, MI) according to the manufacturer’s instructions. A mouse monoclonal antibody to human PCNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as the primary antibody at a dilution of 1:80. To assure the specificity of the immunological reaction, cultured cells were subjected to the same immunoperoxidase method, except that the primary antibody was replaced by nonimmune murine IgG (Miles, Elkhart, IN) at the same dilution as the specific antibody. The replacement of the specific primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining for PCNA.

Immunocytochemical staining was analyzed by two investigators in a blinded fashion without knowledge of the experimental group. The PCNA-positive rate was determined by observing more than 1000 nuclei for each experimental sample and was used for evaluating the proliferating activity of leiomyoma cells.

In situ terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay

In situ labeling of fragmented DNA in cultured leiomyoma cells was performed with the TUNEL assay, using the ApopTag in situ apoptosis detection kit (Intergen Co., Purchase, NY) according to the manufacturer’s protocol for monolayer cultures. Leiomyoma cells were subcultured in two-well glass chamber slides for 120 h and then cultured under serum deprivation conditions for 48 and 96 h in the absence or presence of graded concentrations of CDB-2914. At the termination of cultures, nucleotide-sized DNA fragments were tailed with digoxigenin-deoxy-UTP and then bound with peroxidase-conjugated antidigoxigenin antibodies. The nuclei were counterstained with hematoxylin (Zymed Laboratories, Inc., San Francisco, CA) for determining the TUNEL-positive rate of cultured leiomyoma cells.

Apoptosis of cultured leiomyoma cells was analyzed by two investigators in a blinded fashion without knowledge of the experimental group. All stained nuclei were scored as positive for apoptosis. The TUNEL-positive rate was determined by observing more than 1000 nuclei for each experimental sample.

Western blot analysis for PCNA, Bcl-2, caspase-3, and PARP

Proteins were extracted from cultured leiomyoma cells as described previously (2). At the termination of cultures, cells were lysed at 4 C for 20 min in the presence of a lysis buffer consisting of 150 mM NaCl, 2 mM phenylmethylsulfonylfluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/liter aprotinin, 0.1% sodium dodecyl sulfate, and 50 mM Tris-HCl, pH 7.5. The lysates were subsequently centrifuged at 13,000 x g for 30 min at 4 C, and the supernatants were collected. Protein content in the supernatants was determined by the Bradford assay (31). Each 100-µg aliquot of the protein extracted from cultured leiomyoma cells was electrophoresed on 10% SDS-PAGE under reducing conditions. The proteins were then electrophoretically transferred from gels to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA). The blots were exposed overnight to a mouse monoclonal antibody to PCNA (Santa Cruz Biotechnology, Inc.), a mouse monoclonal antibody to Bcl-2 (Santa Cruz Biotechnology, Inc.), a rabbit polyclonal antibody to caspase-3 that recognizes procaspase and active caspase (Cell Signaling Technology, Inc., Livermore, CA), and a rabbit polyclonal antibody to PARP (Cell Signaling Technology, Inc.) at dilutions of 1:200, 1:200, 1:1000, and 1:1000, respectively. The membranes were incubated for 1 h with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibody (Amersham Biosciences, Arlington Heights, IL) that was diluted at 1:1000 with blocking buffer. The antigen-antibody complexes were detected with the ECL chemiluminescence detection system (Amersham Biosciences). Membranes were visualized by exposure to X-OMAT film (Eastman Kodak Co., Rochester, NY). The radioautograms were then scanned and quantified with ChemiImager 4400 (Astec Co. Ltd., Osaka, Japan).

Statistical analysis

The data were expressed as the mean ± SD. Statistical significance was determined using one- or two-way ANOVA and Tukey’s test with JMP 5.01 software (SAS Institute, Inc., Cary, NC) for Macintosh. Differences with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of CDB-2914 on number of viable cultured leiomyoma cells

The effects of treatment with graded concentrations of CDB-2914 on the number of viable cultured leiomyoma cells were determined by MTT assay. Two-way ANOVA of the indexes for viable cultured leiomyoma cells showed significant effects of CDB-2914 concentrations (P < 0.001) and time of culture (P < 0.01) as well as a significant interaction between time of culture and CDB-2914 concentrations (P < 0.01; Fig. 1Go). In contrast, compared with untreated control cultures, 48-h treatment with CDB-2914 at concentrations higher than 10–7 M significantly (P < 0.05) decreased the number of viable cultured leiomyoma cells, whereas 96-h treatment with CDB-2914 significantly (P < 0.05) decreased the number of viable cells in a dose-dependent manner at concentrations higher than 10–8 M (Fig. 1Go). By contrast, treatment with 10–9 M CDB-2914 did not affect the number of viable cultured leiomyoma cells even after 96 h of treatment.



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  		FIG. 1. Effects of CDB-2914 on the number of viable cultured human leiomyoma cells, as assessed by MTT assay. Compared with untreated control cultures, 48-h treatment with CDB-2914 at concentrations higher than 10–7 M significantly decreased the number of viable cultured leiomyoma cells, whereas 96-h treatment with CDB-2914 significantly decreased the number of viable cells in a dose-dependent manner at concentrations higher than 10–8 M. Results represent the mean ± SD of seven independent experiments performed in triplicate. *, P < 0.05 vs. untreated control cultures.

 
Effects of CDB-2914 on PCNA expression

Figure 2Go represents immunocytochemical staining of PCNA in leiomyoma cells cultured for 48 h (A–D) and 96 h (E–H) under serum-free, phenol red-free conditions in the absence or presence of graded concentrations of CDB-2914. There was no apparent difference in the abundance of PCNA-positive nuclei between cultured leiomyoma cells treated with 10–8 M CDB-2914 for 48 h (Fig. 2BGo) and untreated cells in control cultures for 48 h (Fig. 2AGo), whereas PCNA-positive nuclei in cultured leiomyoma cells treated with either 10–7 M (Fig. 2CGo) or 10–6 M CDB-2914 (Fig. 2DGo) for 48 h were apparently less than those in untreated control cultures for 48 h (Fig. 2AGo). The number of PCNA-positive nuclei in cultured leiomyoma cells treated with either 10–7 M (Fig. 2GGo) or 10–6 M CDB-2914 (Fig. 2HGo) for 96 h was remarkably less than that in untreated control cultures for 96 h (Fig. 2EGo). Replacement of the primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining for PCNA in cultured leiomyoma cell nuclei (data not shown).



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  		FIG. 2. Immunocytochemical staining of PCNA in leiomyoma cells cultured for 48 h (A–D) and 96 h (E–H) under serum-free, phenol-red free conditions in the absence or presence of graded concentrations of CDB-2914. A and E, Untreated; B and F, 10–8 M CDB-2914-treated; C and G, 10–7 M CDB-2914-treated; D and H, 10–6 M CDB-2914-treated. There was no apparent difference in the abundance of PCNA-positive nuclei between cultured leiomyoma cells treated with 10–8 M CDB-2914 for 48 h (B) and untreated cells in control cultures for 48 h (A), whereas PCNA-positive nuclei in cultured leiomyoma cells treated with either 10–7 M (C) or 10–6 M CDB-2914 (D) for 48 h were less apparent than those in untreated control cultures at 48 h (A). The number of PCNA-positive nuclei in cultured leiomyoma cells treated with either 10–7 M (G) or 10–6 M (H) CDB-2914 for 96 h was remarkably less than that in untreated control cultures at 96 h (E). Bars, 5 µm. Original magnification, x400.

 
Figure 3Go shows the PCNA-positive rate of leiomyoma cells cultured in the absence or presence of graded concentrations of CDB-2914 for 48 and 96 h. Two-way ANOVA of the indexes for the PCNA-positive rate showed significant effects of CDB-2914 concentration (P < 0.001) and time of culture (P < 0.001) as well as a significant interaction between time of culture and CDB-2914 concentration (P < 0.001; Fig. 3Go). In contrast, compared with untreated control cultures, treatment with CDB-2914 decreased the PCNA-positive rate of cultured leiomyoma cells in a dose-dependent manner. A significant decrease in the PCNA-positive rate was obtained by 48-h treatment with CDB-2914 at concentrations higher than 10–7 M (P < 0.01) and by 96-h treatment with CDB-2914 at concentrations higher than 10–8 M (P < 0.01).



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  		FIG. 3. The mean percentage of PCNA-positive nuclei of leiomyoma cells cultured in the absence or presence of graded concentrations of CDB-2914, as assessed by immunocytochemical analysis. Compared with untreated control cultures, treatment with CDB-2914 decreased the PCNA-positive rate of cultured leiomyoma cells in a dose-dependent manner. A significant decrease in the PCNA-positive rate was obtained by 48-h treatment with CDB-2914 at concentrations higher than 10–7 M and by 96-h treatment with CDB-2914 at concentrations higher than 10–8 M. Results represent the mean ± SD of seven independent experiments performed in triplicate. *, P < 0.01 vs. PCNA-positive rate in untreated leiomyoma cells in control cultures.

 
Western blot analysis of proteins extracted from leiomyoma cells cultured for 48 h revealed that cultured leiomyoma cells contained immunoreactive PCNA with a molecular mass of approximately 36 kDa. One-way ANOVA of the indexes for PCNA protein content showed significant effects of CDB-2914 concentration (P < 0.001). Compared with untreated control cultures, treatment with CDB-2914 at concentrations higher than 10–7 M significantly (P < 0.05) decreased 36-kDa PCNA expression in cultured leiomyoma cells (Fig. 4Go). There was a significant difference in PCNA expression between 10–7 and 10–6 M CDB-2914 (P < 0.05). In contrast, treatment with 100 ng/ml P4 significantly (P < 0.05) increased 36-kDa PCNA expression compared with untreated control cultures, whereas combined treatment with P4 (100 ng/ml) and CDB-2914 (10–6 M) significantly (P < 0.001) attenuated the P4-induced increase in 36-kDa PCNA protein expression (Fig. 4Go).



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  		FIG. 4. Effects of CDB-2914 on PCNA protein expression in cultured leiomyoma cells at 48 h of treatment, as assessed by Western blot analysis. Compared with untreated control cultures, treatment with CDB-2914 at concentrations higher than 10–7 M significantly decreased 36-kDa PCNA expression in cultured leiomyoma cells. There was a significant difference in PCNA expression between 10–7 and 10–6 M CDB-2914 (P < 0.05). In contrast, treatment with 100 ng/ml P4 significantly increased 36-kDa PCNA expression compared with untreated control cultures, whereas combined treatment with P4 (100 ng/ml) and CDB-2914 (10–6 M) remarkably attenuated the P4-induced increase in 36-kDa PCNA protein expression. Densitometric analysis of PCNA protein levels was performed as described in Materials and Methods. ß-Actin was used to ensure the even loading of each specimen. Results represent the mean ± SD fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. PCNA protein content in untreated leiomyoma cells in control cultures; **, P < 0.001 vs. PCNA protein content in leiomyoma cells treated with P4 alone.

 
Effects of CDB-2914 on the TUNEL-positive rate

Figure 5Go represents the distribution of TUNEL-positive nuclei in leiomyoma cells cultured for 48 h (A–D) and 96 h (E–H) in the absence or presence of graded concentrations of CDB-2914. TUNEL-positive nuclei were labeled in black-brown confined to the nuclei. Compared with untreated control cultures at 48 h (Fig. 5AGo), TUNEL-positive nuclei were more abundant in cultured leiomyoma cells treated with either 10–7 M (Fig. 5CGo) or 10–6 M CDB-2914 (Fig. 5DGo) for 48 h. TUNEL-positive nuclei became more remarkably abundant after 96-h treatment with either 10–7 M (Fig. 5GGo) or 10–6 M (Fig. 5HGo) CDB-2914 relative to those in untreated control cultures for 96 h (Fig. 5EGo). Replacement of the primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining for TUNEL-positive cells in cultured leiomyoma cells (data not shown).



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  		FIG. 5. The distribution of TUNEL-positive nuclei in leiomyoma cells cultured in the absence or presence of graded concentrations of CDB-2914. Isolated leiomyoma cells were cultured for 48 h (A–D) and 96 h (E–H) under serum-free, phenol-red free conditions in the absence or presence of graded concentrations of CDB-2914. TUNEL-positive nuclei were labeled in black-brown. A and E, Untreated; B and F, 10–8 M CDB-2914-treated; C and G, 10–7 M CDB-2914-treated; D and H, 10–6 M CDB-2914-treated. Compared with untreated control cultures at 48 h (A), TUNEL-positive nuclei were more abundant in cultured leiomyoma cells treated with either 10–7 M (C) or 10–6 M (D) CDB-2914 for 48 h and became more remarkably abundant after 96-h treatment with 10–7 M (G) or 10–6 M (H) CDB-2914 relative to those in untreated control cultures at 96 h (E). Bars, 5 µm. Original magnification, x400.

 
Figure 6Go shows the TUNEL positive rate in cultured leiomyoma cells in the absence or presence of graded concentrations of CDB-2914 for 48 and 96 h. Determination of the mean percentage of TUNEL-positive nuclei in cultured leiomyoma cells demonstrated that treatment with CDB-2914 increased the TUNEL positive rate of cultured leiomyoma cells in a dose-dependent manner compared with untreated control cultures. Two-way ANOVA of the indexes for TUNEL-positive rate showed significant effects of CDB-2914 concentrations (P < 0.001) and time of culture (P < 0.001) as well as a significant interaction between time of culture and CDB-2914 concentrations (P < 0.001; Fig. 6Go). In contrast, a significant increase in the TUNEL-positive rate of cultured leiomyoma cells was obtained by 48-h treatment with CDB-2914 at concentrations higher than 10–7 M (P < 0.05) and by 96-h treatment with CDB-2914 at concentrations higher than 10–8 M (P < 0.01).



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  		FIG. 6. Effects of CDB-2914 on the TUNEL-positive rate of cultured leiomyoma cells, as assessed by TUNEL assay. Treatment with CDB-2914 increased the TUNEL-positive rate of cultured leiomyoma cells in a dose-dependent manner compared with untreated control cultures. A significant increase in the TUNEL-positive rate of cultured leiomyoma cells was observed after 48-h treatment with CDB-2914 at concentrations higher than 10–7 M and after 96-h treatment with CDB-2914 at concentrations higher than 10–8 M. Results represent the mean ± SD of seven independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01 (vs. TUNEL-positive rate in untreated leiomyoma cells in control cultures).

 
Time course of the effects of 10–7 M CDB-2914 on procaspase-3, cleaved caspase-3, PARP, and Bcl-2 expression

Figure 7Go represents the time course of procaspase-3, cleaved caspase-3, cleaved PARP, and Bcl-2 protein expression in cultured leiomyoma cells treated with 10–7 M CDB-2914 at 0, 12, 24, and 48 h, as assessed by Western blot analysis. The bottom panel illustrates the changes in the relative intensity of cleaved caspase-3, cleaved PARP, and Bcl-2 protein expression normalized to the respective ß-actin level during the course of treatment with 10–7 M CDB-2914 for 48 h. Compared with untreated leiomyoma cells at 0 h of treatment, cleaved caspase-3 expression was augmented by CDB-2914, with a peak at 12 h of treatment that declined thereafter, whereas cleaved PARP expression was augmented by CDB-2914, with a peak at 24 h of treatment that declined thereafter. In contrast, Bcl-2 protein expression was decreased by CDB-2914 in a time-dependent manner.



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  		FIG. 7. Time course of the effects of 10–7 M CDB-2914 on procaspase-3, cleaved caspase-3, cleaved PARP, and Bcl-2 protein expression in cultured leiomyoma cells, as assessed by Western blot analysis. The bottom panel illustrates the changes in the relative intensity of cleaved caspase-3, cleaved PARP, and Bcl-2 protein expression normalized to the respective ß-actin. Compared with untreated leiomyoma cells at 0 h of treatment, cleaved caspase-3 expression was augmented by CDB-2914, with a peak at 12 h of treatment and a decline thereafter, whereas cleaved PARP expression was augmented by CDB-2914, with a peak at 24 h of treatment and a decline thereafter. In contrast, Bcl-2 protein expression was decreased by CDB-2914 in a time-dependent manner.

 
Effects of graded concentrations of CDB-2914 on cleaved caspase-3 expression

Effects of treatment with graded concentrations of CDB-2914 on cleaved caspase-3 expression in cultured leiomyoma cells at 12 h of treatment were assessed by Western blot analysis (Fig. 8Go). One-way ANOVA of the indexes for cleaved caspase-3 protein content showed significant effects of CDB-2914 concentration (P < 0.001). Although cleaved caspase-3 expression was scarce in leiomyoma cells in untreated control cultures, treatment with CDB-2914 at concentrations higher than 10–8 M significantly (P < 0.05) increased 17-kDa cleaved caspase-3 expression compared with untreated control cultures. There was a significant difference (P < 0.05) in cleaved caspase-3 expression between 10–8 and 10–7 M CDB-2914 and between 10–8 and 10–6 M CDB-2914.



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  		FIG. 8. Effects of treatment with graded concentrations of CDB-2914 on cleaved caspase-3 expression in cultured leiomyoma cells at 12 h of treatment, as assessed by Western blot analysis. Although cleaved caspase-3 expression was scarce in untreated leiomyoma cells in control cultures, treatment with CDB-2914 at concentrations higher than 10–8 M significantly increased 17-kDa cleaved caspase-3 expression compared with untreated control cultures. There was a significant difference (P < 0.05) in cleaved caspase-3 expression between 10–8 and 10–7 M CDB-2914 and between 10–8 and 10–6 M CDB-2914. Densitometric analysis of cleaved caspase-3 levels in cultured leiomyoma cells was performed as described in Materials and Methods. ß-Actin was used to ensure even the loading of each specimen. Results represent the mean ± SD fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. cleaved caspase-3 protein content in untreated leiomyoma cells in control cultures.

 
Effects of graded concentrations of CDB-2914 on cleaved PARP expression

Effects of treatment with graded concentrations of CDB-2914 on cleaved PARP expression in cultured leiomyoma cells at 24 h of treatment were assessed by Western blot analysis (Fig. 9Go). One-way ANOVA of the indexes for cleaved PARP protein content showed significant effects of CDB-2914 concentration (P < 0.001). Compared with untreated control cultures, treatment with CDB-2914 significantly (P < 0.05) increased 89-kDa cleaved PARP expression in a dose-dependent manner. There was a significant difference (P < 0.05) in cleaved PARP expression between 10–8 and 10–7 M CDB-2914, between 10–8 and 10–6 M CDB-2914, as well as between 10–7 and 10–6 M CDB-2914.



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  		FIG. 9. Effects of treatment with graded concentrations of CDB-2914 on cleaved PARP expression in cultured leiomyoma cells at 24 h of treatment, as assessed by Western blot analysis. Compared with untreated control cultures, treatment with CDB-2914 increased 89-kDa cleaved PARP expression in a dose-dependent manner. There was a significant difference (P < 0.05) in cleaved PARP expression between 10–8 and 10–7 M CDB-2914, between 10–8 and 10–6 M CDB-2914, as well as between 10–7 and 10–6 M CDB-2914. Densitometric analysis of cleaved PARP levels in cultured leiomyoma cells was performed as described in Materials and Methods. ß-Actin was used to ensure the even loading of each specimen. PC, Positive control (staurosporin, 10 µg). Results represent the mean ± SD fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. cleaved PARP protein content in untreated leiomyoma cells in control cultures.

 
Effects of graded concentrations of CDB-2914 on Bcl-2 protein expression

Effects of treatment with graded concentrations of CDB-2914 on Bcl-2 protein expression in cultured leiomyoma cells at 48 h of treatment were assessed by Western blot analysis (Fig. 10Go). One-way ANOVA of the indexes for Bcl-2 protein content showed significant effects of CDB-2914 concentration (P < 0.001). Treatment with CDB-2914 at concentrations higher than 10–7 M significantly (P < 0.05) decreased 26-kDa Bcl-2 protein expression in a dose-dependent manner. There was a significant difference (P < 0.05) in Bcl-2 protein expression between 10–8 and 10–7 M CDB-2914, between 10–8 and 10–6 M CDB-2914, as well as between 10–7 and 10–6 M CDB-2914.



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  		FIG. 10. Effects of treatment with graded concentrations of CDB-2914 on Bcl-2 protein expression in cultured leiomyoma cells at 48 h of treatment, as assessed by Western blot analysis. Treatment with CDB-2914 at concentrations higher than 10–7 M significantly decreased 26-kDa Bcl-2 protein expression in a dose-dependent manner. There was a significant difference (P < 0.05) in Bcl-2 protein expression between 10–8 and 10–7 M CDB-2914, between 10–8 and 10–6 M CDB-2914, as well as between 10–7 and 10–6 M CDB-2914. Densitometric analysis of Bcl-2 protein levels in cultured leiomyoma cells was performed as described in Materials and Methods. ß-Actin was used to ensure the even loading of each specimen. Results represent the mean ± SD fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. Bcl-2 protein content in untreated leiomyoma cells in control cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we have demonstrated that the PR modulator CDB-2914 exerts an inhibitory effect on the number of viable cultured leiomyoma cells in a dose-dependent manner. The growth inhibitory effect of CDB-2914 on cultured leiomyoma cells is in agreement with a previous report by Chegini et al. (16), who demonstrated that RU486 and ZK98299 (Onapristone) inhibited [3H]thymidine uptake by cultured leiomyoma cells. Moreover, recent studies have demonstrated that RU486 and ZK98299 can induce growth inhibition in a variety of normal and tumor cells, including endometrial cells (17, 32, 33, 34), granulosa cells (18), breast cancer cells (35, 36, 37, 38, 39), ovarian cancer cells (40), prostate cancer cells (41), and meningioma cells (42). In our study, treatment with CDB-2914 resulted in a decrease in PCNA expression in cultured leiomyoma cells in a dose-dependent manner and reversed the stimulatory effect of P4 on PCNA expression in those cells. These results indicate that CDB-2914 exerts an antiproliferative activity in cultured leiomyoma cells. The antiproliferative activity of CDB-2914 may be associated with cell cycle arrest. Because the expression of PCNA is known to be elevated at the late G1 and S phases of proliferating cells (43), CDB-2914 may cause cell cycle arrest at the G0/G1 phase in cultured leiomyoma cells. Likewise, the antiproliferative activity of RU486 in primate endometrium was reported to be associated with a cell cycle block at the G2-M interphase (34). In addition, RU486 was demonstrated to block PR-expressing human ovarian epithelial carcinoma cell lines in the G0/G1 phase of the cell cycle and reduce the number of cells in the S phase (40). CDB-2914 may also act to modulate the regulation of cell cycle progression.

Another possible explanation of the antiproliferative activity of CDB-12914 is that CDB-2914 may intervene between P4 and growth factors. It has been suggested that cross-talk exists between P4 and growth factors as well as apoptosis regulatory factors in cultured leiomyoma cells (5). Although P4 contributes to the promotion of leiomyoma cell growth through up-regulating epidermal growth factor and the survival through up-regulating Bcl-2 protein expression and down-regulating TNF{alpha} expression in leiomyoma cells, P4 also exerts an inhibitory effect on leiomyoma growth and survival through down-regulating IGF-I expression in those cells. Thus, it is likely that P4 has dual actions on leiomyoma growth depending on the local growth factor conditions around each leiomyoma. Nevertheless, the net effect of P4 on leiomyoma growth seems to favor the mitogenic activity of leiomyoma cells. Actually, the present study demonstrated that CDB-2914 augmented apoptosis of cultured leiomyoma cells in a dose-dependent manner through up-regulating cleaved caspase-3 and cleaved PARP expression and down-regulating Bcl-2 protein expression in those cells. The time-course study demonstrated that cleaved caspase-3 expression was augmented by CDB-2914, with a peak at 12 h of treatment, whereas cleaved PARP expression was augmented by CDB-2914, with a peak at 24 h of treatment. By contrast, Bcl-2 protein expression in those cells was attenuated by CDB-2914 in a time-dependent manner. Because the maximum responses of cleaved caspase-3 and cleaved PARP expression to CDB-2914 treatment were seen at 12 and 24 h of treatment, respectively, the effects of graded doses of CDB-2914 on cleaved caspase-3 expression were evaluated at 12 h of treatment, whereas the effects of graded doses of CDB-2914 on cleaved PARP expression were determined at 24 h of treatment. Compared with untreated control cultures, dose-dependent increases in cleaved caspase-3 and cleaved PARP expression in cultured leiomyoma cells were noted at 12 h of treatment and 24 h of treatment with CDB-2914, respectively.

Apoptotic stimuli have been known to activate 32-kDa procaspase-3 by cleavage to a mature caspase-3 composed of active 17- and 12-kDa subunits (44, 45). The delayed increase in 89-kDa cleaved PARP expression compared with cleaved caspase-3 observed in this study indicates the proteolytic cleavage of PARP by active caspase-3. It has been thought that the cleavage of PARP occurs to prevent the depletion of energy (NAD and ATP) that is required for later stages of apoptosis, and that PARP cleavage serves to prevent futile repair of DNA strand breaks during the apoptotic process (46). Moreover, down-regulation of Bcl-2 protein expression by CDB-2914 may not only diminish its antiapoptotic activity, but could promote the release of cytochrome c into cytosol, thereby further amplifying the caspase cascade. Thus, it is likely that CDB-2914 induces apoptosis of cultured leiomyoma cells by activating caspase cascade.

Substantial evidence indicates that RU486 and ZK98299 can induce apoptosis of a variety of cells, including endometrial cells (17), granulosa cells (18, 19, 20), luteal cells (47, 48), breast cancer cells (35, 38), cervical cancer cells (49), endometrial cancer cells (50), and prostate cancer cells (41, 51, 52, 53). However, only endometrial cells (17) and granulosa cells (20) were cultured in serum-free conditions. All of the cells mentioned above should be cultured under serum-free conditions, because they are believed to be steroid hormone dependent. Furthermore, RU486 is shown to have neuroprotective potential, as evidenced by the observation that RU486 can protect Purkinje cells and hippocampal neurons from the apoptotic process (54, 55). These data suggest that the effects of P4 antagonists on apoptosis may be cell type specific.

Members of the Bcl-2 family are crucial regulators of apoptosis in mammalian cells. The Bcl-2 family includes antiapoptotic proteins such as Bcl-2 and Bcl-xL and proapoptotic proteins such as Bax, Bak, Bad, Bik, Bim, and Bok (30). The relative ratios of antiapoptotic and proapoptotic Bcl-2 family proteins play a major role in determining the ultimate sensitivity or resistance of cells to apoptotic stimuli (30). In the present study, treatment with CDB-2914 down-regulated Bcl-2 protein expression in cultured leiomyoma cells in a dose- and time-dependent manner. Because Bcl-2 prevents mitochondrial permeability pore opening and release of apoptogenic proteins from mitochondria (30), the down-regulation by CDB-2914 of Bcl-2 protein expression in cultured leiomyoma cells may result in the release of cytochrome c and subsequent activation of caspases due to opening of the mitochondrial pore. Schneider et al. (50) reported that RU486 induced apoptosis in three endometrial cancer cell lines (Hec-1A, KLE, and RL95-2). These researchers reported that the proapoptosis gene WAF-1 was significantly increased in Hec-1A and RL95-2 by RU486 treatment compared with control cultures, whereas no change was apparent in KLE cells, and that RU486 did not affect the expression of bcl-2, producing minimal change except for decreased expression in RL95-2 cells. Furthermore, a recent report has demonstrated that RU486 induces apoptosis in human endometrial cells through a marked increase in nuclear factor-{kappa}B-binding activity and subsequent overexpression of bax mRNA and down-regulation of bcl-2 expression (17).

Moreover, several researchers have speculated that TGFß1 may be involved in P4 antagonist-induced apoptosis in cancer cells. RU486 induces cell growth inhibition in both estrogen receptor- and PR-positive MCF-7 human breast cancer cells and human LNCaP prostate cancer cells in association with a significant increase in DNA fragmentation, down-regulation of Bcl-2 protein expression, and up-regulation of TGFß1 protein expression in these cells (38, 41). Recent studies have also demonstrated that RU486 induces apoptosis in estrogen receptor- and PR-negative MDA-231 human breast cancer cells (35) by releasing cytochrome c into the cytoplasm and activating caspase-3, suggesting that the drug-stimulated secretion of TGFß1 is responsible for caspase-3 activity. In uterine leiomyoma cells, RU486 and ZK98299 have been shown to have only limited inhibitory actions on TGFß1 expression (16). Other apoptotic pathways involved in RU486-treated LNCaP and LNCaP C4–2 prostate cancer cells include the TNF{alpha}-related, apoptosis-inducing ligand pathway, which induces apoptosis through interaction with the death domain receptors DR4 and DR5 (51, 53). The biological implication of the TNF{alpha}-related, apoptosis-inducing ligand pathway in the induction of apoptosis in leiomyoma cells remains to be explored.

In conclusion, we have demonstrated that treatment with the PR modulator CDB-2914 inhibits the proliferation of cultured leiomyoma cells by down-regulating PCNA ex-pression and induces apoptosis by up-regulating cleaved caspase-3 and cleaved PARP expression and down-regulating Bcl-2 protein expression in those cells. Additional knowledge about the precise molecular mechanism by which CDB-2914 regulates leiomyoma cell growth and apoptosis will contribute to the potential application of CDB-2914 to the treatment of uterine leiomyoma.


   Acknowledgments
 
We thank Dr. André Ulmann and Ms. Erin E. Gainer of HRA Pharma (Paris, France) for kindly providing us with CDB(VA)-2914 and supporting our research.


   Footnotes
 
This work was supported in part by Grant-in-Aid for Scientific Research 1437053 from the Japanese Ministry of Education, Science, and Culture and by the Ogyaa-Donation Foundation of the Japan Association of Obstetricians and Gynecologists.

First Published Online November 30, 2004

Abbreviations: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide; P4, progesterone; PARP, poly(ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; TUNEL, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling.

Received August 5, 2004.

Accepted November 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Matsuo H, Maruo T, Samoto T 1997 Increased expression of Bcl-2 protein in human uterine leiomyoma and its up-regulation by progesterone. J Clin Endocrinol Metab 82:293–299[Abstract/Free Full Text]
  2. Shimomura Y, Matsuo H, Samoto T, Maruo T 1998 Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma. J Clin Endocrinol Metab 83:2192–2198[Abstract/Free Full Text]
  3. Kurachi O, Matsuo H, Samoto T, Maruo T 2001 Tumor necrosis factor-{alpha} expression in human uterine leiomyoma and its down-regulation by progesterone. J Clin Endocrinol Metab 86:2275–2280[Abstract/Free Full Text]
  4. Rein MS 2000 Advances in uterine leiomyoma research: the progesterone hypothesis. Environ Health Perspect 108(Suppl 5):791–793
  5. Maruo T, Ohara N, Wang J, Matsuo H 2004 Sex steroidal regulation of uterine leiomyoma growth and apoptosis. Hum Reprod Update 10:207–220[Abstract/Free Full Text]
  6. Leonhardt SA, Edwards DP 2002 Mechanism of action of progesterone antagonists. Exp Biol Med 227:969–980[Abstract/Free Full Text]
  7. Eisinger SH, Meldrum S, Fiscella K, Le Roux HD, Guzick DS 2003 Low-dose mifepristone for uterine leiomyomata. Obstet Gynecol 101:243–250[Abstract/Free Full Text]
  8. Kettel LM, Murphy AA, Morales AJ, Yen SSC 1994 Clinical efficacy of the antiprogesterone RU486 in the treatment of endometriosis and uterine fibroids. Hum Reprod 9:116–120
  9. Murphy AA, Morales AJ, Kettel LM, Yen SSC 1995 Regression of uterine leiomyomata to the antiprogesterone RU486: dose-response effect. Fertil Steril 64:187–190[Medline]
  10. Murphy AA, Castellano PZ 1994 RU486: pharmacology and potential use in the treatment of endometriosis and leiomyomata uteri. Curr Opin Obstet Gynecol 6:269–278[Medline]
  11. Murphy AA, Kettel LM, Morales AJ, Roberts VJ, Yen SSC 1993 Regression of uterine leiomyomata in response to the antiprogesterone RU486. J Clin Endocrinol Metab 76:513–517[Abstract]
  12. Attardi BJ, Burgenson J, Hild SA, Reel JR, Blye RP 2002 CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914. Mol Cell Endocrinol 188:111–123[CrossRef][Medline]
  13. Blithe DL, Nieman LK, Blye RP, Stratton P, Passaro M 2003 Development of the selective progesterone receptor modulator CDB-2914 for clinical indications. Steroids 68:1013–1017[CrossRef][Medline]
  14. Gainer EE, Ulmann A 2003 Pharmacologic properties of CDB(VA)-2914. Steroids 68:1005–1011[CrossRef][Medline]
  15. Hild SA, Reel JR, Hoffman LH, Blye RP 2000 CDB-2914: anti-progestational/anti-glucocorticoid profile and post-coital anti-fertility activity in rats and rabbits. Hum Reprod 15:822–829[Abstract/Free Full Text]
  16. Chegini N, Ma C, Tang XM, Williams RS 2002 Effects of GnRH analogues, ‘add-back’ steroid therapy, antiestrogen and antiprogestins on leiomyoma and myometrial smooth muscle cell growth and transforming growth factor-ß expression. Hum Reprod 8:1071–1078
  17. Han S, Sidell N 2003 RU486-induced growth inhibition of human endometrial cells involves the nuclear factor-{kappa}B signaling pathway. J Clin Endocrinol Metab 88:713–719[Abstract/Free Full Text]
  18. Shao R, Markström E, Friberg PA, Johansson M, Billig H 2003 Expression of progesterone receptor (PR) A and B isoforms in mouse granulosa cells: stage-dependent PR-mediated regulation of apoptosis and cell proliferation. Biol Reprod 68:914–921[Abstract/Free Full Text]
  19. Mussche S, D’Herde K 2001 Contribution of progesterone, follicle stimulating hormone and glucocorticoids in survival of serum-free cultured granulosa cell explants. J Endocrinol 169:321–331[Abstract]
  20. Svensson EC, Markström E, Shao R, Andersson M, Billig H 2001 Progesterone receptor antagonists Org31710 and RU486 increase apoptosis in human periovulatory granulosa cells. Fertil Steril 76:1225–1231[CrossRef][Medline]
  21. Siu PM, Bryner RW, Martyn JK, Alway SE 2004 Apoptotic adaptations from exercise training in skeletal and cardiac muscle. FASEB J 18:1150–1152[Abstract/Free Full Text]
  22. Khanna N, Jayaram HN, Singh N 2004 Benzamide riboside induced mitochondrial mediated apoptosis in human lung cancer H520 cells. Life Sci 75:179–190[CrossRef][Medline]
  23. Cohen GM 1997 Caspases: the executioners of apoptosis. Biochem J 326:1–16
  24. Bouchard VJ, Rouleau M, Poirier GG 2003 PARP-1, a determinant of cell survival in response to DNA damage. Exp Hematol 31:446–454[CrossRef][Medline]
  25. Soldani C, Scovassi AI 2002 Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis 7:321–328[CrossRef][Medline]
  26. D’Amours D, Sallmann FR, Dixit V, Poirier GG 2001 Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J Cell Sci 114:3771–3778
  27. Germain M, Affar EB, D’Amours D, Dixit VM, Salvesen GS, Poirier GG 1999 Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. J Biol Chem 274:28379–28384[Abstract/Free Full Text]
  28. Tsujimoto Y, Croce CM 1986 Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA 83:5214–5218[Abstract/Free Full Text]
  29. Schinzel A, Kaufmann T, Borner C 2004 Bcl-2 family members: intracellular targeting, membrane-insertion, and changes in subcellular localization. Biochim Biophys Acta 1644:95–105[Medline]
  30. Reed JC, Jurgensmeier JM, Matsuyama S 1998 Bcl-2 family proteins and mitochondria. Biochim Biophys Acta 1366:127–137[Medline]
  31. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem 72:248–253[CrossRef][Medline]
  32. Chwalisz K, Brenner RM, Fuhrmann UU, Hess-Stumpp H, Elger W 2000 Antiproliferative effects of progesterone antagonists and progesterone receptor modulators on the endometrium. Steroids 65:741–751[CrossRef][Medline]
  33. Murphy AA, Zhou MH, Malkapuram S, Santanam N, Parthasarathy S, Sidell N 2000 RU486-induced growth inhibition of human endometrial cells. Fertil Steril 74:1014–1019[CrossRef][Medline]
  34. Heikinheimo O, Hsiu JG, Gordon K, Kim S, Williams RF, Gibbons WE, Hodgen GD 1996 Endometrial effects of RU486 in primates-antiproliferative action despite signs of estrogen action and increased cyclin-B expression. J Steroid Biochem Mol Biol 59:179–190[CrossRef][Medline]
  35. Liang Y, Hou M, Kallab AM, Barrett JT, El Etreby F, Schoenlein PV 2003 Inhibition of antiproliferation and apoptosis in estrogen receptor negative MDA-231 human breast cancer cells by mifepristone and 4-hydroxytamoxifen combination therapy: a role for TGFß1. Int J Oncol 23:369–380[Medline]
  36. Klijn JGM, Setyono-Han B, Foekens JA 2000 Progesterone antagonists and progesterone receptor modulators in the treatment of breast cancer. Steroids 65:825–830[CrossRef][Medline]
  37. Darro F, Cahen P, Vianna A, Decasestecker C, Nogaret J-M, Leblond B, Chaboteaux C, Ramos C, Pétein M, Budel V, Schoofs A, Pourrias B, Kiss R 1998 Growth inhibition of human in vitro and mouse in vitro and in vivo mammary tumor models by retinoids in comparison with tamoxifen and the RU-486 anti-progestagen. Breast Cancer Res Treat 51:39–55[CrossRef][Medline]
  38. El Etreby MF, Liang Y, Wrenn RW, Schoenlein PV 1998 Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells. Breast Cancer Res Treat 51:149–168[CrossRef][Medline]
  39. Musgrove EA, Lee CSL, Cornish AL, Swarbrick A, Sutherland RL 1997 Antiprogestin inhibition of cell cycle progression in T-47D breast cancer cells is accompanied by induction of the cyclin-dependent kinase inhibitor p21. Mol Endocrinol 11:54–66[Abstract/Free Full Text]
  40. Rose FV, Barnea ER 1996 Response of human ovarian carcinoma cell lines to antiprogestin mifepristone. Oncogene 12:999–1003[Medline]
  41. El Etreby MF, Liang Y, Lewis RW 2000 Induction of apoptosis by mifepristone and tamoxifen in human LNCaP prostate cancer cells in culture. Prostate 43:31–42[CrossRef][Medline]
  42. Matsuda Y, Kawamoto K, Kiya K, Kurisu K, Sugiyama K, Uozumi T 1994 Antitumor effects of antiprogesterones on human meningioma cells in vitro and in vivo. J Neurosurg 80:527–534[Medline]
  43. Robbins BA, De la Vega D, Ogata K, Tan EM, Nakamura RM 1987 Immunohistochemical detection of proliferating cell nuclear antigen in solid human malignancies. Arch Pathol Lab Med 111:841–845[Medline]
  44. Erhardt P, Cooper GM 1996 Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL. J Biol Chem 271:17601–17604[Abstract/Free Full Text]
  45. Nicolson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin T-T, Yu VL, Miller DK 1995 Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37–43[CrossRef][Medline]
  46. Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S, Smulson M 1999 Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. J Biol Chem 274:22932–22940[Abstract/Free Full Text]
  47. Telleria CM, Goyeneche AA, Cavicchia JC, Stati AO, Deis RP 2001 Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy. Endocrine 15:147–155[CrossRef][Medline]
  48. Rueda BR, Hendry IR, Hendry III WJ, Stormshak F, Slayden OD, Davis JS 2000 Decreased progesterone levels and progesterone receptor antagonists promote apoptotic cell death in bovine luteal cells. Biol Reprod 62:269–276[Abstract/Free Full Text]
  49. Kamradt MC, Mohideen N, Vaughan ATM 2000 RU486 increases radiosensitivity and restores apoptosis through modulation of HPV E6/E7 in dexamethasone-treated cervical carcinoma cells. Gynecol Oncol 77:177–182[CrossRef][Medline]
  50. Schneider CC, Gibb RK, Taylor DD, Wan T, Gerçel-Taylor Ç 1998 Inhibition of endometrial cancer cell lines by mifepristone (RU486). J Soc Gynecol Investig 5:334–338[Medline]
  51. Eid MA, Lewis RW, Kumar MV 2002 Mifepristone pretreatment overcomes resistance of prostate cancer cells to tumor necrosis factor {alpha}-related apoptosis-inducing ligand (TRAIL). Mol Cancer Ther 1:831–840[Abstract/Free Full Text]
  52. Liang Y, Eid MA, El Etreby F, Lewis RW, Kumar MV 2002 Mifepristone-induced secretion of transforming growth factor ß1-induced apoptosis in prostate cancer cells. Int J Oncol 21:1259–1267[Medline]
  53. Sridhar S, Ali AA, Liang Y, El Etreby MF, Lewis RW, Kumar MV 2001 Differential expression of members of the tumor necrosis factor {alpha}-related apoptosis-inducing ligand pathway in prostate cancer cells. Cancer Res 61:7179–7183[Abstract/Free Full Text]
  54. Ghoumari AM, Dusart I, El-Etr M, Tronche F, Sotelo C, Schumacher M, Baulieu E-E 2003 Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum. Proc Natl Acad Sci USA 100:7953–7958[Abstract/Free Full Text]
  55. Mccullers DL, Sullivan PG, Scheff SW, Herman JP 2002 Mifepristone protects CA1 hippocampal neurons following traumatic brain injury in rat. Neuroscience 109:219–230[CrossRef][Medline]



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