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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0823
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 10 3949-3957
Copyright © 2007 by The Endocrine Society

Adenovirus-Mediated Delivery of a Dominant-Negative Estrogen Receptor Gene in Uterine Leiomyoma Cells Abrogates Estrogen- and Progesterone-Regulated Gene Expression

Memy H. Hassan, Salama A. Salama, Hossam M. M. Arafa, Farid M. A. Hamada and Ayman Al-Hendy

Department of Obstetrics and Gynecology (M.H.H., S.A.S., A.A.-H.), University of Texas Medical Branch, Galveston, Texas 77555; and Department of Pharmacology and Toxicology (M.H.H., S.A.S., H.M.M.A., F.M.A.H.), Faculty of Pharmacy, Al-Azhar University, 11371 Cairo, Egypt

Address all correspondence and requests for reprints to: Ayman Al-Hendy, M.D., Ph.D., Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0587. E-mail: ayalhend{at}utmb.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Human uterine leiomyomas are very common smooth muscle cell tumors that occur in reproductive-age women and are the leading reason for performing hysterectomies. The present study was conducted to explore the potential mechanism behind the effects exerted by dominant-negative estrogen receptors (DNERs) delivered by adenovirus to leiomyoma cells to ascertain the utility of DNERs as a novel strategy for treatment of uterine fibroids.

Objective and Methods: We investigated the ability of DNER to affect estrogen response element (ERE) activity induced by wild-type estrogen receptor (ER) by using the adenovirus ERE luciferase (Ad-ERE-luc) system in ELT3 cells and the effect of graded doses of DNER (10, 50, and 100 plaque-forming units/cell) on the expression of some selected genes controlling cultured human leiomyoma cell proliferation (cyclin D1, Cox2, PCNA, VEGF, and EGF), apoptosis (Bcl2 and Bax), estrogen metabolism (COMT), and extracellular matrix formation (MMP1) as well as progesterone receptors (A and B) were assessed using Western blot analysis. These genes are all regulated by estrogen and/or progesterone.

Results: DNER has the ability to suppress the ERE luc activity induced by wild-type ER (P < 0.01) and significantly (P < 0.05) reverse the expression of all estrogen- and progesterone-regulated genes in this study.

Conclusions: These results suggest that interruption of the estrogen signaling pathway using DNER results in modulation of both estrogen- and progesterone-regulated genes that control leiomyoma cell apoptosis, proliferation, extracellular matrix formation, progesterone receptors, and estrogen metabolism, which might account for the DNER mechanism of action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UTERINE LEIOMYOMAS (fibroids) are benign smooth muscle tumors originating from the myometrium. Leiomyomas are estrogen-dependent neoplasms (1), whereas the effects of estrogens are mediated by the estrogen receptor (ER). Both subtypes, ER{alpha} and ERß, have been found in fibroids (2, 3). Substantial evidence indicates an increase in estrogen-binding sites in leiomyomas, and this increase may account for their enhanced sensitivity to estrogen (4, 5, 6). Estrogens induce cell proliferation in leiomyoma tissues by stimulating progression through the G1 phase of the cell cycle (7). Induction of cyclin D1 expression is a critical feature of the mitogenic action of estrogen (8). Other potential mechanisms by which estrogen stimulates the proliferation and growth of uterine leiomyomas involve the regulation of the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) (9), which is highly expressed in leiomyoma tissues compared with the normal adjacent myometrium (10).

Progesterone is implicated in leiomyoma growth (5, 11, 12, 13). Several studies have demonstrated that progesterone receptor (PR) is up-regulated in uterine leiomyomas compared with adjacent normal myometrium at mRNA and protein levels (6, 14, 15, 16). Progesterone functions as a ligand-activated transcription factor to regulate the expression of specific sets of target genes in leiomyoma cells, such as the antiapoptotic protein Bcl-2, proliferating cell nuclear antigen (PCNA), and epidermal growth factor (EGF) (17, 18).

Interestingly, the expression of PRs is regulated by estrogen (19, 20, 21). Therefore, it is anticipated that interception of ER signaling will have a dual action on leiomyoma cells, a direct effect by disrupting the expression of estrogen-regulated growth factors as well as an indirect effect via down-regulation of PRs and their downstream effectors. Dominant-negative ERs (DNERs) are ER mutants that are unable to activate transcription but can suppress the transcriptional activity of the wild-type ER (both ER{alpha} and ERß) when they are coexpressed in the same cells (22).

Our previous study demonstrated that transfection of leiomyoma cells with the adenovirus vector expressing DNER severely inhibited cell proliferation and resulted in a marked increase in the number of apoptotic cells (23).

In this study, we examined the effects of Ad-DNER transfection on the expression of genes regulated by estrogen, progesterone, or both as a potential mechanism by which Ad-DNER exerts its effects on leiomyoma cells to ascertain the utility of DNERs as a novel treatment of uterine fibroids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant adenovirus

The Ad-DNER mutant, ER1–536 (under the cytomegalovirus promoter used in this work), has been described previously (23, 24). An adenoviral reporter vector carrying two copies of the estrogen-responsive element (ERE) upstream of luciferase reporter (Ad-ERE-luc) (kindly provided by Dr. Eun J. Lee, Northwestern University, Chicago, IL) was used to investigate transcriptional activity of estrogen receptor. Adenovirus expressing a marker gene coding for bacterial ß-galactosidase (Ad-LacZ) was a kind gift from Dr. Savio Woo (Mount Sinai School of Medicine, New York, NY) and was used as a negative control. Large-scale production of adenovirus vectors was performed as we described previously (25) with a typical batch yield of 2 x 1010 plaque-forming units (pfu)/ml.

Cell cultures and transfection with adenovirus vectors

The ELT3 cell line, a rat leiomyoma cell line that expresses both ER and PR (26), was a kind gift from Dr. Cheryl Walker (University of Texas, M. D. Anderson Cancer Center, Smithville, TX). ELT3 was maintained at 37 C in 5% CO2/air in DMEM, with 10% fetal bovine serum. Human leiomyoma tissues were collected according to the policies of the Institutional Review Board at the University of Texas Medical Branch and used to establish primary human leiomyoma cells, as described previously (23, 27). The immortalized human uterine leiomyoma cell line (huLM), which expresses both ERs and PRs, was a gift from Dr. Darlene Dixon (National Institute of Environmental Health Sciences, Research Triangle Park, NC). The cells were cultured and maintained as described previously (28). The transfection with adenoviral vectors was conducted as we have described previously (23, 29, 30). The maximum transfection efficiency (95–100%) was attained at a multiplicity of infection (MOI) of 100 pfu/cell for ELT3 cells and 10 pfu/cell for human leiomyoma cells after 48 h using Ad-LacZ (data not shown).

Luciferase reporter assays in ELT3 leiomyoma cell line

The effect of Ad-DNER on the transcriptional activity of the ER was assayed using an estrogen-responsive reporter in a viral vector (Ad-ERE-luc), as we have described previously (6). Briefly, ELT3 leiomyoma cells were transfected overnight in 12-well plates with Ad-ERE-Luc 100 pfu/cell, and the next day the media were replaced with transfection media containing increasing amounts (0, 1, 10, 50, 100, and 200 pfu/cell) of Ad-DNER. Fresh medium with estradiol (10–8 M) was added, and incubation was continued for 48 h. Luciferase activities were determined using luciferase enzyme assay systems, according to the supplier’s protocol (Promega, Madison, WI). The luciferase activity was normalized against the number of cells per well calculated by measuring fluorescence of Hoechst 33258 dye (23).

Western blot analysis of ER- and/or PR-regulated gene expression in cultured human uterine leiomyoma cells

Both primary and immortalized human leiomyoma cells were plated separately in 10-cm culture dishes at a density of 5 x 105 cells per plate. The following day, they were transfected with Ad-DNER at 0, 10, 50, and 100 pfu/cell for 5 h, as described previously (23). After the addition of fresh medium, the cells were incubated for 48 h with 10–8 M of 17ß-estradiol (E2). Cells were washed twice with PBS, and whole-cell lysates were prepared with lysis buffer [25% glycerol, 0.5 M NaCl, 1.5 mM MgCl2, 20 mM HEPES (pH 7.9), 1 mM phenylmethylsulfonyl fluoride, 0.2 mM EDTA, 25 mM NaF, and protease inhibitor cocktail tablets; Roche Molecular Biochemicals, Indianapolis, IN]. Equal amounts of protein (40 µg) were resolved by SDS-PAGE gel. The membranes were blocked with 5% nonfat milk in Tris-buffered Tween 20 buffer for 1.5 h and then incubated overnight at 4 C with the indicated primary antibodies. After three washes in Tris-buffered Tween 20 buffer, immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL). Bands were detected with X-Omat film (Eastman Kodak Co., Rochester, NY). The radioautograms were scanned and quantified with Flurochem/{alpha} Ease FC imaging system (Alpha Innotech, San Leandro, CA). The relative intensities of each protein level were normalized with corresponding ß-actin values. These experiments were repeated with three different cultured cell specimens from human primary leiomyoma cells and the human immortalized leiomyoma cell line with similar results, and the reported results are typical.

Statistical analysis

The data are presented as mean ± SE from three experiments with similar results. Group differences were analyzed using one-way ANOVA followed by Tukey test as a post-ANOVA multiple comparison test; P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Ad-DNER on transcriptional activity of the endogenous ER in rat leiomyoma cells

To investigate whether the Ad-DNER affects the ability of the endogenous ER to activate an ERE and, hence, gene expression, ELT3 rat leiomyoma cells were transfected with either Ad-ERE-luc alone (control) or in combination with ascending doses of Ad-DNER (1, 10, 50, 100, and 200 pfu/cell). Transfection with Ad-DNER suppressed reporter activities in ELT3 rat leiomyoma cells (Fig. 1Go). Ad-DNER at MOI of 10, 50, 100, and 200 pfu/cell suppressed the Ad-ERE-luc reporter activity by 30, 92, 96, and 97%, respectively (P < 0.001) compared with the Ad-DNER at MOI l pfu/cell. Interestingly, transfection of ELT3 rat leiomyoma cells with Ad-DNER at MOI 1 pfu/cell increased ERE-luc transactivation compared with untransfected control cells (Fig. 1Go). Recently we have confirmed these findings in a telomerase immortalized human leiomyoma cell line using Ad-LacZ as an additional negative control (data not shown).


Figure 1
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  		FIG. 1. Effect of Ad-DNER on transcriptional activity of endogenous ER of rat uterine leiomyoma cells (ELT3 cell line). The ER transcriptional activity was assayed using Ad-ERE-luc, as described in Materials and Methods, and the relative light units were normalized to the cell number. Results are plotted as mean ± SE of three independent experiments. P < 0.01 was considered a significant difference from control (a) or from Ad-DNER 1 pfu/cell (b).

 
Effect of Ad-DNER on the expression of apoptosis-regulating genes in human primary leiomyoma cells

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 proapoptotic proteins, such as Bax (31). When infected with Ad-DNER at MOI of 10, 50, and 100 pfu/cell, primary human leiomyoma cells demonstrated a dose-dependent decrease in the expression of the antiapoptotic Bcl-2 protein, whereas the proapoptotic Bax protein does not seem to be affected (Fig. 2AGo). Moreover, the Bax/Bcl-2 expression ratio was increased in a dose-dependent manner compared with the control (P < 0.05) (Fig. 2BGo).


Figure 2
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  		FIG. 2. Effect of Ad-DNER on the expression of apoptosis-regulating genes in cultured primary human leiomyoma cells as assessed by Western blot. Ad-DNER decreases antiapoptotic protein Bcl-2 and did not affect proapoptotic BAX protein expression (A), which increases the expression ratio of Bax to Bcl2 proteins (B). The cultured cells were treated for 48 h with transfection media alone (control) or an ascending amount of Ad-DNER (10, 50, and 100 pfu/cell). Densitometric analysis was performed as described in Materials and Methods. Data are presented as a fold of the control value and as the mean ± SE of at least three independent experiments. a, Significant difference from control culture at P < 0.05.

 
Transfection with Ad-DNER down-regulates the expression of proliferation-regulating genes

Dixon et al. (32) concluded that a higher rate of cell proliferation plays a pivotal role in uterine leiomyoma growth. In this study, we evaluated the expression of an endogenous marker of cell proliferation, PCNA (33, 34), as well as cyclin D1 and COX2 that strictly regulate the cell cycle (35, 36). Western blot analysis of proteins extracted from cultured leiomyoma cells (primary and immortalized), treated with Ad-DNER at 10, 50, and 100 pfu/cell for 48 h revealed that leiomyoma cells contained immunoreactive PCNA with a molecular mass of approximately 36 kDa, as shown in Fig. 3AGo. PCNA protein contents decreased significantly in a dose-dependent manner compared with untreated control cells (P < 0.05).


Figure 3
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  		FIG. 3. Ad-DNER decreases the expression of proliferation-regulating genes PCNA (A), cyclin D1 (B and C), and COX2 (D and E) in both human primary (PL) and immortalized (huLM) leiomyoma cells, respectively, as assessed by Western blot analysis. Densitometric analysis was performed as described in Materials and Methods. Data are presented as a fold of the control value and as the mean ± SE of at least three independent experiments. P < 0.05 was considered significant compared with untreated control cultures (a) or Ad-LacZ 100 pfu/cell-treated cultures (b).

 
Specific bands for cyclin D1 and COX2 were detected at approximately 38 and 71 kDa, respectively, in human primary and immortalized leiomyoma cells, and Ad-DNER caused a significant (P < 0.05) and dose-dependent decrease in these bands’ densities when compared with either untreated or Ad-LacZ-treated cells (Fig. 3Go, B–E).

Effect of Ad-DNER on the expression of growth factors in human leiomyoma cells (VEGF and EGF)

The VEGF protein was detected at 22 kDa, and the band density was significantly decreased (P < 0.001) in dose-dependent manner in both primary and immortalized human leiomyoma cells at higher doses of Ad-DNER (Fig. 4AGo). Also, Western blot analysis with a monoclonal antibody to EGF revealed that the cultured immortalized leiomyoma cells contained immunoreactive EGF with a molecular mass of approximately 7 kDa and that the transfection with Ad-DNER resulted in a remarkable decrease in the level of its expression in a dose-dependent manner (Fig. 4BGo)


Figure 4
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  		FIG. 4. Western blot analysis for the effect of Ad-DNER on expression of growth factors. A and B, VEGF (A) and EGF (B) in both human primary (PL) and immortalized (huLM) leiomyoma cells. Densitometric analysis was performed as described in Materials and Methods. Data are presented as a fold of control value and as the mean ± SE. This figure is representative of three separate experiments. a, Significant difference from control at P < 0.05; b, significant difference from Ad-LacZ 100 pfu/cell at P < 0.05.

 
Effect of Ad-DN-ER on the expression of progesterone receptors in human leiomyoma cells

Treatment of both primary and immortalized human leiomyoma cells with Ad-DNER (10, 50, and 100 pfu/cell) resulted in a significant decrease in the expression of both PRs (A and B) (Fig. 5Go, A and B). Densitometric evaluation revealed that the decrease was more impressive in PR-A (Fig. 5Go, A and B).


Figure 5
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  		FIG. 5. Effect of Ad-DNER on the expression of PR-A and -B in both cultured human primary leiomyoma (PL) cells (A) and immortalized human leiomyoma (huLM) cells (B). a, Significant difference from control at P < 0.05; b, significant difference from Ad-LacZ 100 pfu/cell at P < 0.05.

 
Transfection with Ad-DNER down-regulates the expression of the estrogen-metabolizing gene (COMT) in primary human leiomyoma cells

To demonstrate the effect of Ad-DNER on the pathway controlling estrogen metabolism, we evaluated the COMT expression in primary human leiomyoma cells after transfection with different MOI of Ad-DNER (10, 50, and 100 pfu/cell). The protein expression of both soluble and membrane-bound COMT was significantly decreased in a dose-dependent fashion compared with untransfected control cells (P < 0.05) (Fig. 6AGo).


Figure 6
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  		FIG. 6. Effect of Ad-DNER on the expression of COMT, soluble (S) and membrane-bound (MB) isoforms, in primary human leiomyoma cells (A) and MMP1 in both cultured human immortalized (huLM) and primary leiomyoma (PL) cells (B). Densitometric analysis was performed as described in Materials and Methods. Data are presented as a fold of the control value and as the mean ± SE of at least three independent experiments. a, Significant difference from control at P < 0.05; b, significant difference from Ad-LacZ 100 pfu/cell at P < 0.05.

 
Transfection with Ad-DNER up-regulates the expression of matrix metalloproteinase 1 (MMP1) protein expression in human leiomyoma cells

A specific band for MMP1 was detected at about 55 kDa in both immortalized and primary human leiomyoma cells (Fig. 6BGo). Transfection with Ad-DNER (10, 50, and 100 pfu/cell) resulted in significant augmentation of MMP1 protein expression in the immortalized human leiomyoma cell line (P < 0.01) and a remarkable increase in the primary cell type in a dose-dependent manner relative to control cultured cells (Fig. 6BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that estrogen and progesterone play a vital role in leiomyoma growth (37). Brandon et al. (4, 14) demonstrated increased expression of ERs and PRs in uterine leiomyomas compared with the adjacent normal myometrium. It is, therefore, conceivable that there is currently great interest in exploring approaches to exploit these steroid receptors as potential therapeutic targets for uterine leiomyomas. One approach includes the use of DNERs to inactivate endogenous ERs (22). Recent reports from our laboratory demonstrated that transfection of human uterine leiomyoma cells with Ad-DNER was efficient at 10 pfu/cell and resulted in suppression of cell proliferation and induction of apoptosis, as well as regression of in vivo lesions in nude mice (23). In this report, we wanted to examine the mechanisms by which ER abrogation induced by Ad-DNER transfection leads to such changes in uterine leiomyoma cells. Here, we have illustrated that Ad-DNER transfection suppresses the global ability of wild-type ER to transactivate ERE in rat leiomyoma cells using an ERE-luciferase probe (Fig. 1Go). We have also demonstrated that adenovirus-mediated ER gene targeting using DNER is effective in abrogating the expression of several estrogen- and/or progesterone-regulated genes, representing various cellular pathways that have been reported to be altered in uterine leiomyoma, such as apoptosis represented by Bcl-2 and Bax; proliferation represented by PCNA, cyclin D1, COX2, VEGF, EGF; estrogen metabolism represented by COMT; and extracellular matrix represented by MMP1 as well as progesterone receptors (PR-A and PR-B) as potential mechanisms of action (Figs. 2–6GoGoGoGoGo).

Due to adenovirus receptor heterogeneity, the human adenovirus can optimally transfect the rat ELT3 cells only at 100 pfu/cell, whereas in human leiomyoma cells, optimal transfection can be achieved at a much lower MOI (10 pfu/cell) (23). Based on that, it is not surprising that in ELT3 cells, we saw a significant decrease in luc activity only at 50 pfu/cell or higher (Fig. 1Go). The marked nonspecific increase in luc at the 1 pfu/cell concentration was unexpected and may reflect a partial agonist initial intrinsic effect of the DNER on ERE at the lower dose. This conclusion is plausible because we excluded any potential effect of the adenovirus back bone on ERE by repeating the experiment on huLM cells exploiting Ad-lacZ as additional negative control (data not shown). Similar findings have been reported previously (22, 24). It is worth noting that most effects exerted by estrogen are mediated through ERE, and most estrogen-regulated genes exhibit ERE sites in their promoter regions (38, 39). This suggests that Ad-DNER treatment would have a major effect on the expression of most estrogen-regulated genes.

The development and progress of leiomyoma is believed to be regulated, in part, by the genes controlling the apoptosis and proliferation processes (32, 40). Apoptosis is inhibited by the Bcl-2/Ced-9 family of proteins (41). The Bcl-2 gene is overexpressed in many tumors (42, 43), including leiomyoma (5, 44, 45). Estrogen is known to up-regulate Bcl-2 transcription in ER-positive MCF-7 and T47D human breast cancer cells (43, 46, 47), whereas the induction of apoptosis by RU 486, an antiprogesterone agent, in both ER- and PR-positive MCF, is accompanied by a decrease in Bcl-2 (48, 49). Perillo et al. (50) revealed that estrogen induction is mediated by two ERE motifs present in the Bcl-2-coding region. Consistent with these findings, we have shown that transfection of Ad-DNER decreased estrogen-induced Bcl-2 expression (Fig. 2AGo). Because Bcl-2 prevents mitochondrial permeability, pore opening, and release of apoptogenic proteins from mitochondria (31), its down-regulation by Ad-DNER may result in release of cytochrome c and subsequent activation of caspases triggering the apoptotic cascade (31, 51, 52).

However, the ratio of Bcl-2 to Bax, rather than the absolute level of either protein, may determine the sensitivity to apoptosis (53). In this study, Ad-DNER increased this ratio in a dose-dependent manner (Fig. 2BGo). Taken together, Ad-DNER seems to induce apoptotic changes in all tested human leiomyoma cells, a process mediated, at least in part, via increasing the Bax to Bcl-2 expression ratio.

We have also evaluated the expression of PCNA protein, which is considered to be an endogenous marker for proliferation (32, 54). In addition to an essential role of PCNA in DNA replication, other reports suggest the involvement of PCNA in DNA excision repair (55, 56). Shimomura et al. (12) demonstrated that E2 as well as P4 increased the PCNA-labeling index of cultured leiomyoma cells and augmented the PCNA protein expression in those cells. Furthermore, a higher PCNA-labeling index, indicating higher proliferation rate, was observed in leiomyoma tissues relative to the adjacent normal myometrial tissues throughout the menstrual cycle (12, 32). In our study, treatment of cultured leiomyoma cells with Ad-DNER resulted in a dose-dependent decrease in PCNA expression (Fig. 3AGo); these results indicate that DNER exerts antiproliferative activity in cultured leiomyoma cells. The antiproliferative activity of Ad-DNER 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 (54). Thus, we evaluated the effect of such treatment on the expression of certain cell cycle-regulating genes, cyclin D1 and COX2. It is widely accepted that cell proliferation is strictly regulated by cell cycle control mechanisms, which depend on the activities of different cyclins, e.g. cyclin D1 and COX 2 (35, 57, 58, 59). Estrogen induces expression of cyclin D1 in many tumors, including leiomyomas, and it is implicated in its pathogenesis and growth (7, 60). In the current study, DNER decreased the expression of cyclin D1 at all tested dose levels, which may indicate that DNER has the ability to slow or even arrest the cell cycle progression and, therefore, proliferation of leiomyoma cells (Fig. 3Go, B and C). Likewise, COX2 expression was shown to be up-regulated by E2 in the myometrium (61), and it has been linked to tumorigenesis and angiogenesis (62, 63). It is generally believed that overexpression of COX2 leads to proliferative and/or angiogenic effects through prostaglandin-dependent (64, 65) or -independent mechanisms (66). Ad-DNER treatment again decreased the expression of the COX2 gene at the protein level (Fig. 3Go, D and E) and, thus, contributed to the inhibition of cell cycle progression and the killing of the leiomyoma cells.

In the present study, we also provide evidence that Ad-DNER is capable of decreasing the expression of growth factors in leiomyoma, e.g. EGF and VEGF (Fig. 4Go, A and B). VEGF is critical for the initiation and maintenance of the angiogenic response, a major process by which new blood vessels are formed in the adult (67). Uterine leiomyomas were reported to express the VEGF protein (10, 68, 69), which is regulated by estrogen as well (9) and has been implicated in leiomyoma growth (68, 69). Our study demonstrates that Ad-DNER decreases the expression of the VEGF protein in a dose-dependent manner in leiomyoma cells (Fig. 4AGo), which could conceivably have adverse effects on leiomyoma growth. The expression of immunoreactive EGF proteins is thought to be involved in the autocrine/paracrine regulation of leiomyoma growth (70, 71, 72, 73). A potential role for EGF in the regulation of leiomyoma growth was also suggested by Lumsden et al. (74), who demonstrated that GnRH agonist caused shrinkage of uterine leiomyoma size in conjunction with a remarkable reduction in uterine EGF-binding sites. In connection with this, we have noticed in the present study that DNER also has the capability of decreasing the expression of EGF in leiomyoma cells (Fig. 4BGo).

Shimomura et al. (12) reported that progesterone induced both PCNA and EGF protein expression in cultured leiomyoma cells. Considering our results regarding the ability of Ad-DNER to decrease the expression of both of these genes, we wondered whether the interruption of the estrogen signaling pathway by DNER may indirectly affect progesterone-regulated genes as well. We decided to directly evaluate the effect of Ad-DNER transfection on the expression of PRs in leiomyoma cells. Several investigators have shown an increased concentration of both PR isoforms (PR-A and PR-B) in leiomyoma tissue compared with adjacent myometrium (14). Furthermore, treatment with medroxyprogesterone acetate was associated with an increase in mitotic activity in fibroid tissue relative to the adjacent myometrial tissue (75, 76). Progesterone has also been linked to increased expression of proliferation markers Ki-67 and PCNA in the leiomyoma compared with the normal myometrium (12, 14). Ad-DNER treatment did, indeed, decrease the expression of both PR-A and PR-B in a dose-dependent manner (Fig. 5Go, A and B), which, in turn, would be expected to affect progesterone-regulated gene expression. This result may, in part, explain the down-regulation of EGF and PCNA after Ad-DNER transfection in leiomyoma cells.

We have also proposed that higher COMT levels wash away the antiestrogenic 2-hydroxyestrogen metabolite and, thus, contribute to initiation and progression of leiomyoma lesions (6). Catechol-O-methyltransferase (COMT) metabolizes many endobiotics, including catecholestrogens (77). We have reported recently that the gene for COMT is highly expressed in human leiomyoma tissues compared with adjacent normal myometrium (78). We have also demonstrated that its expression is up-regulated by progesterone in leiomyoma cells both in protein analysis as well as promoter-reporter studies (78). In the present study, we demonstrated that the expression of COMT in leiomyoma cells was decreased in response to Ad-DNER treatment (Fig. 6AGo). This is most likely a downstream effect secondary to the Ad-DNER-induced down-regulation of PRs. Such an effect on COMT expression might lead to the accumulation of the antiestrogenic 2-hydroxyestrogen metabolite, a COMT substrate, which may lead to a hypoestrogenic milieu inside the leiomyoma cells and, therefore, augment the potential therapeutic effects of Ad-DNER. We have recently described a similar effect using COMT inhibitors on rat and human leiomyoma cells (6).

Uterine leiomyomas are characterized by rapid growth and expansion of the extracellular matrix within the boundaries of a growing mesenchymal tumor (79). MMP1 is an enzyme responsible for the degradation of extracellular matrix, and its level is decreased by estrogen (79, 80). The Ad-DNER treatment up-regulated MMP1 expression in a dose-dependent manner (Fig. 6BGo). This will have paramount importance in the ability of DNER to shrink preexisting in vivo leiomyoma tumors, which consist mostly of extracellular matrices (23).

In conclusion, our data demonstrate that using adenovirus coding for a DNER is an effective way to abrogate estrogen- and/or progesterone-regulated gene expression in human leiomyoma cells. These findings confirm the potential value of this strategy, which may lead to an alternative nonsurgical approach for treatment of uterine leiomyomas.


   Acknowledgments
 
We are grateful to Ye Wang for her excellent technical help.


   Footnotes
 
This work was supported by National Institutes of Health/National Institute of Child Health and Human Development Grant 1 R01 HD046228-01 (to A.A.-H.).

Disclosure Summary: The authors (M.H.H., S.A.S., H.M.M.A., F.M.A.H., and A.A.-H.) have nothing to declare.

First Published Online July 17, 2007

Abbreviations: COMT, Catechol-O-methyltransferase; DNER, dominant-negative ER; E2, 17ß-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; MMP1, matrix metalloproteinase 1; MOI, multiplicity of infection; PCNA, proliferating cell nuclear antigen; pfu, plaque-forming units; PR, progesterone receptor; VEGF, vascular endothelial growth factor.

Received April 12, 2007.

Accepted July 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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