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RU486-Induced Growth Inhibition of Human Endometrial Cells Involves the Nuclear Factor-B Signaling Pathway

Division of Research, Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322

Address all correspondence and requests for reprints to: Neil Sidell, Ph.D., Department of Gynecology and Obstetrics, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, Georgia 30322. E-mail: nsidell{at}emory.edu.

	Abstract

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Previous studies indicated that the antiprogestin RU486 could directly inhibit the growth of normal and malignant human endometrial cells. However, the mechanism by which this occurs is poorly understood. In this study we explore further details of endometrial cell growth regulation by RU486. Gel shift assays using the endometrial cell line EM42 demonstrated that RU486, at concentrations ranging from 20–100 µM, significantly stimulated the cellular binding activity of the nuclear transcription factor nuclear factor-B (NF-B) while having little effect on activating protein-1 (AP-1) binding. This effect on NF-B binding was blocked in the presence of the NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC). The data also showed that the activity of RU486 on NF-B binding correlated with the ability of this compound to induce apoptosis of EM42 cells. To investigate a cause and effect relationship between these two phenomena, we evaluated the effects of RU486 treatment on the expression of two apoptosis-related genes, bax and bcl-2, known to be regulated through NF-B binding on their promoter. RT-PCR demonstrated that RU486 significantly induced bax mRNA levels, while suppressing mRNA of bcl-2. Alteration of these genes by RU486 was inhibited in the presence of 100 µM PDTC. Correspondingly, PDTC antagonized the ability of RU486 to inhibit the growth and induce apoptosis of EM42 cells. This study demonstrates that the inhibition of growth and apoptosis of human endometrial cells by RU486 involves stimulation of NF-B binding with subsequent modulation of apoptosis regulatory genes bax and bcl-2.

	Introduction

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RU486, MIFEPRISTONE, HAS been shown to have significant clinical potential in the medical management of endometriosis. Women with laparoscopically proven disease were treated with RU486 and were found to have a decrease in pelvic pain and an improvement in the revised American Fertility Society score (1). Although the mechanism by which RU486 modulates endometrial and endometriotic tissue growth is poorly understood, we (2) and others (3) have shown that it has direct inhibitory effects on cell growth as well as apoptosis-inducing activity (4).

The decision of cells to undergo apoptosis is controlled by external signals in combination with an autonomous genetic program. Cell death is regulated at several intracellular checkpoints by the actions of different members of the bcl-2 gene family (5). The bcl-2 oncogene, which was first identified in human B cell tumors (6), protects against apoptosis and is the founding member of this family. Another member of the family is bax, which promotes apoptosis and encodes a protein that can form homodimers or heterodimers with bcl-2 (7). In human epithelial endometrial cells, bcl-2 predominates at the end of the follicular phase and is low or absent in the secretory phase, when electron microscopic analysis shows apoptotic cells (8). These studies suggested that bcl-2 might be involved in protection against apoptosis during the proliferative phase of the menstrual cycle.

The transcription factor NF-B site has been identified in the promoters of bcl-2 and bax as well as numerous other genes that play a role in cell growth regulation and proliferation (9). Reports indicate that activation of NF-B can induce apoptosis in some cells (10, 11), whereas inhibition of NF-B during the resolution of inflammation helps protract the inflammatory response and prevent apoptosis (12). This knowledge has prompted the present study to investigate the mechanism by which endometrial cell growth is inhibited by RU486. Our results demonstrate that RU486 rapidly induces apoptosis in human endometrial cells. This effect occurs via a NF-B-dependent mechanism and appears to involve the two apoptosis-regulating genes, bcl-2 and bax.

	Materials and Methods

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Cell culture and chemicals

The human endometrial epithelial cell line EM42 was established from benign proliferative endometrium from a patient undergoing hysterectomy for leiomyomata and was originally a gift from Dr. N. Desai (University MacDonald Women’s Hospital, Case Western Reserve University, Cleveland, OH) (13). Cells were grown in RPMI 1640 medium (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 µg amphotericin (complete medium) as previously described (14). RU486 and phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO) were dissolved in dimethylsulfoxide (0.2% of final volume). Pyrrolidine dithiocarbamate (PDTC; Sigma-Aldrich) was dissolved in PBS buffer solution. TACs Apoptotic DNA Laddering Kit was obtained from R\|[amp ]\|D Systems, Inc. (Minneapolis, MN). Poly(dI-dC) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ); NF-B and activating protein-1 (AP-1) consensus oligonucleotides were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); the 5' DNA Terminus Labeling System was purchased from Life Technologies, Inc. (Gaithersburg, MD); [-³²P]deoxy-ATP was purchased from PerkinElmer (Boston, MA). All RT-PCR kit components were obtained from PerkinElmer (PE Applied Biosystems, Foster City, CA). All other chemicals were purchased from Sigma-Aldrich unless otherwise indicated.

EMSA

Nuclear protein extracts from EM42 cells were prepared for EMSA as described previously (15). The protein content of the nuclear extract was determined using the bicinchoninic acid protein assay kit (Sigma-Aldrich) according to the manufacturer’s protocol. EMSA experiments were performed using double-stranded oligonucleotides comprising the consensus sequences (underlined) for NF-B (5'-AGTTGAGGGGACTTTCCCAGGC) and AP-1 (5'-GCTTGATGACTCAGCCGGAA). The binding site for AP-1 comprised the phorbol ester-responsive element. Oligonucleotides were end labeled with [-³²P]deoxy-ATP using T4 polynucleotide kinase as recommended by the manufacturer. In the mutated NF-B oligonucleotide, the B motif was changed to GGcGACTTTCCC. For mutated AP-1 oligonucleotide, the B motif was changed to TGACTtg. Nuclear proteins (3 µg for NF-B, 5 µg for AP-1) from control and treated cells were incubated with the ³²P-labeled oligonucleotide probe under binding conditions [10 mM HEPES (pH 7.9), Tris-HCl (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 12% (vol/vol) glycerol, and 2 µg poly(dI-dC)] for 20 min at room temperature in a final volume of 20 µl. For cold competition, a 100-fold excess of the respective unlabeled consensus oligonucleotides (AP-1 and NF-B) was added with the probe. The same amount of mutated oligonucleotides added with the probe was used as another control. All of these used the same binding conditions as described above. After binding, protein-DNA complexes were electrophoresed on a native 4.5% polyacrylamide gel at 120 V using 1x Tris-glycine buffer (10x Tris-glycine: 30.28 g Tris base, and 142.7 g glycine, 3.92 g EDTA, with H₂O added up to 1 liter, pH 8.5). Each gel was then dried and subjected to autoradiography at -80 C for up to 72 h.

Cell growth

EM42 cells (3 x 10⁵ cells/well) were seeded in six-well plates and incubated with vehicle control, 20 µM RU486, 100 µM PDTC, or 100 µM PDTC for 1 h, followed by 20 µM RU486, then treated for a total of 1–4 d. On d 1, 2, 3, and 4, the cells were harvested by trypsinization, and viable cells were counted using a hemocytometer by trypan blue exclusion.

Detection of DNA fragmentation

Internucleosomal degradation of genomic DNA due to activation of endogenous endonuclease is one feature that occurs during apoptosis. The release of oligonucleosome-associated DNA fragments in this process results in a DNA ladder when analyzed by agarose gel electrophoresis. Detecting internucleosomal DNA fragmentation and displaying DNA laddering using TACS Apoptotic DNA Laddering Kits (R\|[amp ]\|D Systems, Inc.) were used to evaluate apoptotic cell death. Procedures for DNA isolation and detection followed the protocol provided by the product supplier. After running it in a 1.5% TreviGel 500 gel (wt/vol) in 1x Tris-acetate, DNA was visualized with ethidium bromide staining under a UV transilluminator and photographed.

RT-PCR

Total RNA was prepared from cultured EM42 cells using TRIzol reagent (Life Technologies, Inc.). The sequences of PCR primers synthesized by Genosys (The Woodlands, TX) were: for bax: sense, 5'-CCAGCTCTGAACAGATCATG-3'; antisense, 5'-AGCTCCATATTGCTATCCAG-3'; for bcl-2: sense, 5'-CAATCAAAGCCAAGCAGA-3'; antisense, 5'-CCAAACATCCAGAGACAA-3'; for glyceraldehyde-3-phosphate-dehyrogenase (GAPDH): sense, 5'-CCATGGAGAAGGCTGGGG-3'; antisense, 5'-CAAAGTTGTCATGGATGACC-3', according to published data (14, 16). The RT-PCR was carried out as previously described (14). The samples were first denatured at 95 C for 30 sec, followed by 32 PCR cycles, each with temperature variations as follows: 95 C for 30 sec, 60 C for 30 sec, and 72 C for 1 min. The last cycle was followed by an additional extension incubation of 7 min at 72 C. Analysis of amplicons was visualized on 1% agarose gel containing 0.2 µg/µl ethidium bromide and were visualized under UV transilluminator. The densitometric analysis of PCR products was performed using the computer software (Quantity One, Bio-Rad Laboratories, Inc., Hercules, CA) and a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc.), and was standardized by GAPDH. The ratio of bax or bcl-2/GAPDH product bands in the control group was considered 100%. Values for the treatment group bax or bcl-2/GAPDH ratio were given as a percentage of the control. A 100-bp ladder (Life Technologies, Inc.) was used as a size standard.

Statistical analysis

All experiments were repeated a minimum of three times. All gel-shift assays and RT-PCR data are expressed as the mean ± SE. The data in most figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined by t test (two-tailed) comparison between two groups in a dataset. The asterisks shown in the figures indicate significant differences between experimental groups and the corresponding control condition (see figure legends).

	Results

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RU486 increases NF-B activation

Previous studies showed that RU486 could inhibit endometrial cell growth (2, 3). To elucidate the mechanism(s) responsible for the RU486 inhibition, we performed gel mobility shift assays to determine whether this inhibition might occur via NF-B- or/and AP-1-dependent mechanisms. As shown in Fig. 1, culturing EM42 cells with 100 µM RU486 for 24 h caused a marked induction of NF-B binding compared with the control. We also evaluated for RU486 regulation of AP-1 binding activity. As shown in Fig. 2, 100 µM RU486 only slightly stimulated AP-1 binding compared with the control group. As a result of competition assays, specific bands for NF-B (Fig. 1, lane 2) and AP-1 (Fig. 2, lane 4) were attenuated by a 100-fold molar excess of unlabeled respective oligonucleotides. There were no specific bands when the NF-B (Fig. 1, lane 5) or AP-1 (data not shown) probes were mutagenized. These results all indicated that both bindings were specific.

View larger version (25K): [in this window] [in a new window]   Figure 1. RU486 increases the binding activity of NF-B. EM42 cells were cultured with solvent control (Con), 10 µM phorbol 12-myristate 13-acetate, or 100 µM RU486 as indicated for 24 h. After homogenization of the cells, nuclear extracts were prepared, and 3 µg nuclear protein were incubated with a 32P-labeled oligonucleotide containing a canonical NF-B-binding site. The protein DNA complexes were assessed by EMSA. A 100-fold molar excess of unlabeled (Cold) NF-B oligonucleotide incubated with the 32P-labeled probe was used for competition. NIH-3T3 cells were used as a positive control (3T3). The NF-B probe was mutagenized to show specificity of binding (Mut). NS, Nonspecific binding; FP, free probe (A). The bar graph (B) represents the mean ± SE relative NF-B band density of at least three independent experiments.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effects of RU486 on AP-1 binding activation. EM42 cells were cultured with solvent control (Con) or 100 µM RU486 as indicated for 24 h. After homogenization of the cells, nuclear extracts were prepared, and 5 µg nuclear protein were incubated with a 32P-labeled oligonucleotide containing a canonical AP-1-binding site. The protein-DNA complexes were assessed by EMSA. A 100-fold molar excess of unlabeled (Cold) AP-1 oligonucleotide incubated with the 32P-labeled probe was used for competition. NIH-3T3 was used as a positive control (3T3). NS, Nonspecific binding; FP, free probe (A). The bar graph (B) represents the mean ± SE relative AP-1 band density of at least three independent experiments.

Previous reports have demonstrated that RU486 can induce apoptosis in various cell types and is considered to be mediated through several mechanisms (17, 18). Figure 3 shows that RU486 also induced apoptosis of EM42 endometrial cells; a distinct oligosomal ladder was observed 3 d after treatment with RU486 (100 µM). This effect was negligible in cells treated with vehicle (Fig. 3, lane 1).

View larger version (25K): [in this window] [in a new window]   Figure 3. RU486-induced apoptosis. Cells were treated with solvent control (lane 1) or 100 µM RU486 (lane 2) for 72 h. Cellular DNA was isolated as described in Materials and Methods. The electrophoretic pattern of DNA isolated after RU486 treatment was shown by running in a 1.5% TreviGel 500 gels in 1x TAE. The data shown are representative of three experiments.

Having determined that RU486 treatment of EM42 cells induced NF-B-binding activity and apoptosis of the cells, we addressed the question of a possible link between these two phenomena. The NF-B signaling cascade is involved in the induction of apoptosis in a variety of circumstances through its regulation of the apoptosis-related genes, bcl-2 and bax (19). In many cells, bax promotes apoptosis by opposing the action of bcl-2 (20). Inhibition of NF-B has been shown to reduce bax expression in vivo (12). Thus, using RT-PCR, we assessed the ability of RU486 to alter bax and bcl-2 mRNA in EM42 cells. As shown in Fig. 4, culturing cells with 100 µM RU486 for 3 d significantly increased bax mRNA, whereas it down-regulated bcl-2 gene expression compared with the control.

View larger version (16K): [in this window] [in a new window]   Figure 4. RU486 increases bax mRNA while decreasing bcl-2 mRNA levels. Total RNA was isolated from EM42 cells cultured for 72 h with 100 µM RU486 or solvent control as indicated, then subjected to RT-PCR analysis using bax, bcl-2, and GAPDH primers. GAPDH mRNA expression was used as an internal control for normalization purposes (A). The bar graph (B) represents the mean ± SE bax/GAPDH or bcl-2/GAPDH DNA products of at least three independent experiments. *, Significant differences compared with the vehicle control.

Inhibition of NF-B activity suppresses RU486 effects on bax and bcl-2 gene expression

To establish a cause and effect relationship between RU486 induction of apoptosis and NF-B binding, we used PDTC as a general inhibitor of NF-B to prevent NF-B-mediated transcriptional activation (21). PDTC, at a concentration of 100 µM alone, decreased NF-B-binding activity, but had no significant effect on bax or bcl-2 gene expression. In contrast, pretreatment with this compound for 1 h, followed by treatment with RU486 for another 24 h, significantly blocked RU486 induction of NF-B binding compared with the control or PDTC alone (Fig. 5) and also significantly antagonized RU486 up-regulation of bax and down-regulation of bcl-2 gene expression (Fig. 6). These findings indicate that RU486-induced changes in bax and bcl-2 expression in EM42 endometrial cells are NF-B dependent and support the concept that activation of NF-B plays a necessary role in the clinical efficacy of RU486 in endometrial cell growth inhibition.

View larger version (17K): [in this window] [in a new window]   Figure 5. PDTC suppresses RU486-induced activation of NF-B. EM42 cells were treated with vehicle control (Con), 100 µM RU486, 100 µM PDTC, or 100 µM PDTC for 1 h followed by 100 µM RU486 as indicated for 24 h. After homogenization of the cells, nuclear extracts were prepared, and 3 µg nuclear protein were incubated with a 32P-labeled NF-B probe. The protein-DNA complexes were assessed by EMSA. FP, Free probe (A). The bar graph (B) represents the mean ± SE relative NF-B binding density of at least three independent experiments. *, Significant differences compared with the vehicle control; **, significant difference compared with RU486 treatment alone.

View larger version (27K): [in this window] [in a new window]   Figure 6. Inhibition of NF-B activation suppresses RU486 effects on bax and bcl-2 mRNA levels. Total RNA was isolated from EM42 cells cultured for 72 h with solvent control (Con), 100 µM RU486, 100 µM PDTC, or 100 µM PDTC for 1 h followed by 100 µM RU486 as indicated, then subjected to RT-PCR analysis using bax, bcl-2, and GAPDH primers. GAPDH mRNA expression was used as an internal control for normalization purposes. The bar graph represents the mean ± SE bax/GAPDH or bcl-2/GAPDH DNA products of at least three independent experiments. *, Significant differences compared with the vehicle control; **, significant difference compared with RU486 treatment alone.

Inhibition of NF-B by PDTC suppresses RU486-induced growth inhibition and apoptosis of EM42 cells

To confirm that transcriptional activation by NF-B is directly involved in the cellular effects of RU486, we assessed whether treatment with PDTC also antagonized RU486-mediated growth inhibition and induction of apoptosis. Figure 7 shows that this was indeed the case; growth inhibition of EM42 cells by RU486 was partially reversed by pretreating the cells with PDTC for 1 h before adding RU486 for various time periods (Fig. 7A). After 4 d of RU486 treatment, there were very few viable cells remaining in the RU486 cultures, whereas the number of cells cultured with RU486 plus PDTC were approximately 60% of control value. Figure 7B demonstrates that PDTC also reduced the appearance of distinct DNA laddering caused by RU486, an indication that apop-tosis was also inhibited. PDTC alone showed little effect on cell growth or apoptosis.

View larger version (22K): [in this window] [in a new window]   Figure 7. PDTC inhibits the effects of RU486 on cell growth and apoptosis. A, EM42 cells were treated as indicated in the bar graph legend with vehicle control (Con), 20 µM RU486, 100 µM PDTC, or 100 µM PDTC for 1 h followed by 20 µM RU486, then treated for a total of 1–4 d. Viable cell numbers were determined by trypan blue staining. Results represent the mean ± SE of three independent experiments, each performed in duplicate. B, Cells were treated with 100 µM PDTC alone (lane 1), 100 µM RU486 alone (lane 3), or 100 µM PDTC for 1 h followed by 100 µM RU486 (lane 2) for a total of 72 h. Cellular DNA was then isolated as described in Materials and Methods. The electrophoretic patterns of DNA isolated after treatment were shown by running in a 1.5% TreviGel 500 gels in 1x TAE.

	Discussion

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NF-B, one of the early response transcription factors, has been shown to play a pivotal role in the regulation of genes that are involved in the cascade of events leading to cellular apoptosis (20, 26). Matsushita et al. (27) demonstrated that up-regulation of NF-B in endothelial cells stimulated apoptosis by 75%. Haddad and Land (19) found that a decrease in NF-B activity through stimulation of I-B expression inhibited apoptosis-related events in both human umbilical vein endothelial cells and calf pulmonary artery endothelial cells. In the present study we demonstrated that RU486 can markedly increase NF-B-binding activity in human endometrial cells, but has little effect on AP-1. Thus, although NF-B and AP-1 have shown synchronous regulation by a variety of hormone receptor agonists and antagonists (28) as well as antioxidants (29), it is apparent that they are differentially regulated by RU486. Our finding of a link between RU486 induction of apoptosis and the ability of this compound to up-regulate NF-B is consistent with studies demonstrating a similar link in human leukemic T cells (30).

In contrast to our results demonstrating up-regulation of NF-B binding by RU486 in EM42 endometrial cells, we showed in a previous study that RU486 could retard nuclear binding of NF-B induced by the linoleate oxidative product 13-hydroperoxide octadecadienoic acid (13-HPODE) in the mouse macrophage cell line RAW (31). Although the reason(s) for the opposite effects on NF-B activity of RU486 in EM42 vs. RAW cells is unknown, conflicting results have similarly been reported in disparate cells types with other agents. Thus, the natural peroxisome proliferator-activated receptor- (PPAR) agonist 15-deoxy-^12,14-prostaglandin J₂ has been shown to induce binding of NF-B in activated human T lymphocytes (32), while markedly inhibiting NF-B activity in human monocytes (33). One reason for the cell type specificity in the effects of agents on NF-B could be that different signaling pathways may be evoked in the different cell systems. Thus, in the case of 15-deoxy-^12,14-prostaglandin J₂, while its inhibition of NF-B in monocytes has been shown to be mediated through PPAR (33), induction of NF-B in activated T cells does not occur through this signaling pathway (32). As 13-HPODE may also function as a PPAR ligand (34), inhibition of 13-HPODE-induced NF-B activity by RU486 in monocytes is likely to be PPAR dependent. On the other hand, there is no evidence of such dependence in the action of RU486 on NF-B in EM42 cells.

Cellular programming resulting in apoptosis involves several genes, including bcl-2 and bax. There are conflicting reports about the effects of RU486 on these two genes. Several studies (18, 35) indicated that RU486 down-regulated bcl-2 gene expression but had little effect on bax. In contrast, other reports have shown that RU486 can induce bcl-2 while showing no effect on bax expression (36). The fact that the upstream promoter region of these two genes contains NF-B-binding sites (37, 38) together with the suggested physiological roles of members of the bcl-2 gene family in regulating apoptosis of normal cycling endometrial cells (8) prompted us to investigate a possible association among RU486-induced regulation of apoptosis, NF-B, and bcl-2/bax expression in EM42 cells. The results showed that RU486 treatment significantly increased bax while reducing bcl-2 expression. The direction of both of these RU486-mediated changes is consistent with the well characterized roles of these genes in affecting apoptosis (39). We also determined that the specific NF-B inhibitor, PDTC, prevented RU486 up-regulation of bax and down-regulation of bcl-2 in these cells, and that growth inhibition and DNA fragmentation induced by RU486 treatment can be suppressed by cotreatment with PDTC. These findings confirm that the induction of NF-B binding by RU486 plays an essential role in regulating apoptosis-related events in EM42 cells. The down-regulation of bcl-2 expression resulting from RU486 induction of NF-B activity is surprising. For most genes, binding of NF-B proteins to promoter sites results in activation, rather than suppression, of transcription (40). However, conditions giving rise to negative regulation by NF-B have also been described. For example, Matsushita et al. (27) showed that activation of NF-B by hypoxia down-regulated bcl-2 expression in endothelial cells, with a resulting increase in apoptosis. In another study, it was shown that PDTC significantly attenuated the hypoxia-induced apoptosis of endothelial cells by blocking NF-B activation and down-regulation of bcl-2 (41). Similar protection against apoptosis by PDTC has been reported in colon cancer cells (42), although in various other cell systems blockade of NF-B by PDTC was demonstrated to induce apoptosis (43, 44). As such, the effects of increased NF-B binding by RU486 or other compounds on cell growth and viability are clearly cell type specific and may depend on the resultant effects of a variety of apoptosis-related genes whose expression is regulated by NF-B trans-activation as well as the quality of regulation (positive or negative) induced by NF-B on each individual gene.

Taken together, our study showed that the ability of RU486 to regulate endometrial cell growth is at least partially due to promotion of cellular apoptosis. This effect corresponds to a marked increase in NF-B-binding activity and subsequently to overexpression of bax and down-regulation of bcl-2. As RU486 or related compounds are being increasingly considered in the medical management of endometriosis, studies of its mechanism of action in the reproductive tract need to be aggressively pursued.

	Acknowledgments

 

	Footnotes

 
This work was supported by NIH Research Grant P01-HD-35276.

Abbreviations: AP-1, Activating protein-1; GAPDH, glyceraldehyde-3-phosphate-dehyrogenase; 13-HPODE, 13-hydroperoxide octadecadienoic acid; NF-B, nuclear factor-B; PDTC, pyrrolidine dithiocarbamate; PPAR, peroxisome proliferator-activated receptor-.

Received June 4, 2002.

Accepted October 17, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kettel LM, Murphy AA, Morales AJ, Yen SS 1998 Preliminary reports on the treatment of endometriosis with low-dose mifepristone (RU 486). Am J Obstet Gynecol 178:1151–1156[CrossRef][Medline]
2. 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]
3. Prange-Kiel J, Rune GM, Wallwiener D, Kiesel L 2000 Inhibition of proliferation and differentiation by RU 486 in human endometrial stromal and epithelial cells in vitro. Exp Clin Endocrinol Diabetes 108:275–281[CrossRef][Medline]
4. 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]
5. Wang Q, Maloof P, Wang H, Fenig E, Stein D, Nichols G, Denny TN, Yahalom J, Wieder R 1998 Basic fibroblast growth factor downregulates Bcl-2 and promotes apoptosis in MCF-7 human breast cancer cells. Exp Cell Res 238:177–187[CrossRef][Medline]
6. Reed JC 1997 Bcl-2 family proteins: regulators of apoptosis and chemoresistance in hematologic malignancies. Semin Hematol 34(4 Suppl 5):9–19
7. Gallaher BW, Hille R, Raile K, Kiess W 2001 Apoptosis: live or die-hard work either way. Horm Metab Res 33:511–519[CrossRef][Medline]
8. Tao XJ, Tilly KI, Maravei DV, Shifren JL, Krajewski S, Reed JC, Tilly JL, Isaacson KB 1997 Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax. J Clin Endocrinol Metab 82:2738–2746[Abstract/Free Full Text]
9. Chen F, Castranova V, Shi X 2001 New insights into the role of nuclear factor-B in cell growth regulation. Am J Pathol 159:387–397[Abstract/Free Full Text]
10. Southall MD, Isenberg JS, Nakshatri H, Yi Q, Pei Y, Spandau DF, Travers JB 2001 The platelet-activating factor receptor protects epidermal cells from tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand-induced apoptosis through an NF-B-dependent process. J Biol Chem 276:45548–45554[Abstract/Free Full Text]
11. Huang Y, Johnson KR, Norris JS, Fan W 2000 Nuclear factor-B/IB signaling pathway may contribute to the mediation of paclitaxel-induced apoptosis in solid tumor cells. Cancer Res 60:4426–4432[Abstract/Free Full Text]
12. Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA 2001 Possible new role for NF-B in the resolution of inflammation. Nat Med 7:1291–1297[CrossRef][Medline]
13. Desai NN, Kennard EA, Kniss DA, Friedman CI 1994 Novel human endometrial cell line promotes blastocyst development. Fertil Steril 61:760–766[Medline]
14. Han SW, Greene ME, Pitts J, Wada RK, Sidell N 2001 Novel expression and function of peroxisome proliferator-activated receptor (PPAR) in human neuroblastoma cells. Clin Cancer Res 7:98–104[Abstract/Free Full Text]
15. Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract/Free Full Text]
16. Kuwano K, Hagimoto N, Tanaka T, Kawasaki M, Kunitake R, Miyazaki H, Kaneko Y, Matsuba T, Maeyama T, Hara N 2000 Expression of apoptosis-regulatory genes in epithelial cells in pulmonary fibrosis in mice. J Pathol 190:221–229[CrossRef][Medline]
17. Svensson EC, Markstrom E, Shao R, Andersson M, Billig H 2001 Progesterone receptor antagonists Org 31710 and RU 486 increase apoptosis in human periovulatory granulosa cells. Fertil Steril 76:1225–1231[CrossRef][Medline]
18. 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]
19. Haddad JJ, Land SC 2000 The differential expression of apoptosis factors in the alveolar epithelium is redox sensitive and requires NF-B (RelA)-selective targeting. Biochem Biophys Res Commun 271:257–267[CrossRef][Medline]
20. Barkett M, Gilmore TD 1999 Control of apoptosis by Rel/NF-B transcription factors. Oncogene 18:6910–6924.[CrossRef][Medline]
21. Keates S, Hitti YS, Upton M, Kelly CP 1997 Helicobacter pylori infection activates NF-B in gastric epithelial cells. Gastroenterology 113:1099–1109[CrossRef][Medline]
22. Schneider CC, Gibb RK, Taylor DD, Wan T, Gercel-Taylor C 1998 Inhibition of endometrial cancer cell lines by mifepristone (RU 486). J Soc Gynecol Invest 5:334–338[Medline]
23. Brenner RM, Slayden OD 1994 Oestrogen action in the endometrium and oviduct of rhesus monkeys during RU486 treatment. Hum Reprod 9(Suppl 1): 82–97
24. Parthasarathy S, Morales AJ, Murphy AA 1994 Antioxidant: a new role for RU-486 and related compounds. J Clin Invest 94:1990–1995
25. van den Berg HW, Lynch M, Martin JH 1993 The relationship between affinity of progestins and antiprogestins for the progesterone receptor in breast cancer cells (ZR-PR-LT) and ability to down-regulate the receptor: evidence for heterospecific receptor modulation via the glucocorticoid receptor. Eur J Cancer 29A:1771–1775
26. Yamamoto Y, Gaynor RB 2001 Therapeutic potential of inhibition of the NF-B pathway in the treatment of inflammation and cancer. J Clin Invest 107:135–142[Medline]
27. Matsushita H, Morishita R, Nata T, Aoki M, Nakagami H, Taniyama Y, Yamamoto K, Higaki J, Yasufumi K, Ogihara T 2000 Hypoxia-induced endothelial apoptosis through nuclear factor-B (NF-B)-mediated bcl-2 suppression: in vivo evidence of the importance of NF-B in endothelial cell regulation. Circ Res 86:974–981[Abstract/Free Full Text]
28. Auphan N, Ghosh S, Flavell RA, Schmitt-Verhulst AM 1999 Differential requirements for NF-B and AP-1 trans-activation in response to minimal TCR engagement by a partial agonist in naive CD8 T cells. J Immunol 163:5219–5227[Abstract/Free Full Text]
29. Shrivastava A, Aggarwal BB 1999 Antioxidants differentially regulate activation of nuclear factor-B, activator protein-1, c-jun amino-terminal kinases, and apoptosis induced by tumor necrosis factor: evidence that JNK and NF-B activation are not linked to apoptosis. Antioxid Redox Signal 1:181–191[Medline]
30. Ramdas J, Harmon JM 1998 Glucocorticoid-induced apoptosis and regulation of NF-B activity in human leukemic T cells. Endocrinology 139:3813–3821[Abstract/Free Full Text]
31. Sidell N, Han SW, Parthasarathy S 2002 Regulation and modulation of abnormal immune responses in endometriosis. Ann NY Acad Sci 955:159–173[Abstract/Free Full Text]
32. Harris SG, Smith RS, Phipps RP 2002 15-Deoxy-^12,14-PGJ₂ induces IL-8 production in human T cells by a mitogen-activated protein kinase pathway. J Immunol 168:1372–1379[Abstract/Free Full Text]
33. Ji JD, Cheon H, Jun JB, Choi SJ, Kim YR, Lee YH, Kim TH, Chae IJ, Song GG, Yoo DH, Kim SY, Sohn J 2001 Effects of peroxisome proliferator-activated receptor- (PPAR-) on the expression of inflammatory cytokines and apoptosis induction in rheumatoid synovial fibroblasts and monocytes. J Autoimmun 17:215–221[CrossRef][Medline]
34. Van den Heuvel JP 1999 Peroxisome proliferator-activated receptors: a critical link among fatty acids, gene expression and carcinogenesis. J Nutr 129:575S–580S[Abstract/Free Full Text]
35. 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]
36. Critchley HO, Tong S, Cameron ST, Drudy TA, Kelly RW, Baird DT 1999 Regulation of bcl-2 gene family members in human endometrium by antiprogestin administration in vivo. J Reprod Fertil 115:389–395[Abstract]
37. Tsukahara T, Kannagi M, Ohashi T, Kato H, Arai M, Nunez G, Iwanaga Y, Yamamoto N, Ohtani K, Nakamura M, Fujii M 1999 Induction of Bcl-x_L expression by human T-cell leukemia virus type 1 Tax through NF-B in apoptosis-resistant T-cell transfectants with Tax. J Virol 73:7981–7987[Abstract/Free Full Text]
38. Bentires-Alj M, Dejardin E, Viatour P, Van Lint C, Froesch B, Reed JC, Merville MP, Bours V 2001 Inhibition of the NF-B transcription factor increases Bax expression in cancer cell lines. Oncogene 20:2805–2813[CrossRef][Medline]
39. Mullauer L, Gruber P, Sebinger D, Buch J, Wohlfart S, Chott A 2001 Mutations in apoptosis genes: a pathogenetic factor for human disease. Mutat Res 488:211–231[CrossRef][Medline]
40. Matthews JR, Hay RT 1995 Regulation of the DNA binding activity of NF-B. Int J Biochem Cell Biol 27:865–879[CrossRef][Medline]
41. Aoki M, Nata T, Morishita R, Matsushita H, Nakagami H, Yamamoto K, Yamazaki K, Nakabayashi M, Ogihara T, Kaneda Y 2001 Endothelial apoptosis induced by oxidative stress through activation of NF-B: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension 38:48–55[Abstract/Free Full Text]
42. Matroule JY, Carthy CM, Granville DJ, Jolois O, Hunt DW, Piette J 2001 Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide: a methylester photosensitization. Oncogene 20:4070–4084[CrossRef][Medline]
43. Chae H, Chae S, Park N, Bang B, Cho S, Kim J, Kim H, Kim H, Lee Z, Kim H 2001 Pyrrolidine dithiocarbamate inhibits serum-induced NF-B activation and induces apoptosis in ROS 17/2.8 osteoblasts. Int Immunopharmacol 1:255–263[CrossRef][Medline]
44. Santos-Silva MC, Sampaio de Freitas M, Assreuy J 2001 Killing of lymphoblastic leukemia cells by nitric oxide and taxol: involvement of NF-B activity. Cancer Lett 173:53–61[CrossRef][Medline]

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