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Regulation of Apoptosis in Uterine Leiomyomata¹

Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center (K.D.B., K.K., S.R.H., R.F.-Y., D.T., C.W.), Smithville, Texas 78957; Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences (J.C.B.), Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Dr. Kevin D. Burroughs, Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957.

	Abstract

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Tumors developing from hormone-dependent tissues, such as the breast and prostate, have been successfully treated in the clinic by methods of hormone ablation, and the resulting tumor regression has been shown to occur at least in part by the process of apoptosis. The growth of leiomyomas arising from uterine smooth muscle cells is similarly modulated by circulating steroid hormones and has been associated with periods of increased estrogen secretion. The inhibition of ovarian hormone production by endocrine therapy often results in the regression of these tumors, but the role of apoptosis in this process has not been elucidated. Using cell lines derived from the Eker rat model of uterine leiomyoma, we have investigated the mechanism of growth inhibition by estrogen deprivation. Estrogen-depleted medium and the antiestrogen tamoxifen significantly reduced cell numbers in culture and arrested cell proliferation, but did not induce apoptosis. However, the presence of an intact apoptotic pathway was demonstrated in these cells by serum starvation. In vivo data were in agreement with in vitro results, which showed that tamoxifen treatment does not change the apoptotic rate of leiomyoma tissues. Therefore, growth modulation of leiomyomas by hormone deprivation occurs via mechanisms independent of apoptosis, indicating a fundamental difference in the response of leiomyomas to hormone deprivation from that of tumors of the breast and prostate. These data suggest that creation of a hypoestrogenic milieu within leiomyomas reduces tumor volume without inducing a concomitant increase in the rate of apoptosis, which may be responsible for the limited effectiveness of currently available medicinal therapies.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE SUCCESSFUL use of endocrine therapy for the treatment of hormone-sensitive cancers has led to intense investigation into the mechanisms of tumor growth inhibition and regression by these methods. This research has resulted in the recognition of the importance of the role of programmed cell death in the response of these tumors to hormone ablation. When grown as nude mouse xenografts, human mammary and prostatic adenocarcinoma cell lines undergo apoptosis upon hormone withdrawal (1, 2), and the use of antiestrogens in vivo also leads to inhibition of tumor growth and induction of apoptosis in human breast cancer cell lines (3). In vitro assays of hormone sensitivity appear to reliably reflect the ability of tumor cells to undergo hormonally regulated apoptosis. When treated by hormone withdrawal or with the antiestrogens tamoxifen or ICI 182,780, MCF-7 cell cultures exhibit characteristics of programmed cell death (3, 4, 5). Therefore, the ability of endocrine therapy to induce apoptosis in these and other tumor models offers an attractive means of treatment that can be assayed quickly and inexpensively using in vitro techniques.

The ability of estrogens to modulate the growth of uterine leiomyomas, commonly referred to as fibroids, has been well documented. Leiomyomas are typically diagnosed during the reproductive years, can increase in size during pregnancy, and can regress after the menopause (6). However, estrogen is not mitogenic for mature normal myometrial cells, suggesting that estrogen responsiveness has been altered in fibroids relative to that in normal adult myometrium (7, 8). This increased growth response of fibroids to estrogen has commonly been attributed to a hypersensitive state of tumor cells to this hormone (6, 9, 10). Consistent with this hypothesis, estrogen receptors have been shown to be overexpressed in myomas with respect to adjacent myometrium (11, 12, 13). In addition, the rate of estrogen metabolism by tumor cells has been shown to be altered, possibly creating a local hyperestrogenic environment within the tumor (14).

Current nonsurgical management of leiomyomata relies on reducing circulating levels of ovarian hormones with the use of GnRH agonists (15, 16). Such strategies result in the regression of fibroids during treatment by creating a hypoestrogenic state in the individual through desensitization of signaling pathways within the hypothalamic-pituitary axis (17). However, serious side-effects associated with the use of GnRH analogs are the bone loss and increase in blood lipid levels that occur due to the reduced levels of circulating estrogens (18, 19), and it is the increased risk for early-onset osteoporosis and cardiovascular disease that precludes the long term use of these drugs. After the cessation of therapy, regrowth of tumors usually occurs when normal hormonal fluctuations involved in the menstrual cycle are reestablished (20). As a result, GnRH agonists have been limited to the role of preoperative adjuncts that facilitate surgical removal of the tumor or uterus by reducing its size and blood flow (21, 22, 23, 24).

The Eker rat model of uterine leiomyoma previously described by Everitt et al. (25) and Howe et al. (26) allows a unique opportunity for in vitro/in vivo studies of the mechanisms of hormone-stimulated growth of leiomyomas and their response to endocrine manipulation. The antiestrogen tamoxifen, used extensively in therapeutic trials for the prevention and treatment of breast cancer, is thought to act primarily by competitively binding to the estrogen receptor and preventing estradiol from exerting its growth stimulatory effects (27, 28). Initial experiments in the Eker rat to assess the hormonal responsiveness of fibroids, using tamoxifen as a prototypical antiestrogen, demonstrated the ability of this drug to inhibit the proliferation of cell lines derived from Eker uterine leiomyomas (ELT lines) (29). Additionally, tamoxifen exhibited a growth inhibitory effect in vivo by increasing tumor latency and decreasing mean tumor size in nude mice injected with a tumorigenic representative cell line, ELT-3 (29).

Due to the observed regression of fibroids in response to hypoestrogenism and the sensitivity of other hormone-responsive neoplasms to the induction of apoptosis upon hormone withdrawal, we have investigated the mechanism by which estrogen withdrawal inhibits the growth of leiomyomata. Because GnRH agonists inhibit the secretion of gonadotropins and ovarian hormones at the level of the pituitary gland, in vitro studies of leiomyoma growth necessitate the use of alternative methods for inducing hypoestrogenic conditions. Previously, our laboratory has examined the effect of tamoxifen on this tumor due to tamoxifen’s widespread use in clinical trials. This work demonstrated that tamoxifen acts as an antagonist, inhibiting the growth of uterine leiomyoma cell lines in vitro and in nude mice (29). We report here that the proliferation of uterine leiomyoma cells is sensitive to the availability of estrogen in culture, and withdrawal of this hormone or treatment with the antiestrogen tamoxifen inhibits cell proliferation independent of an apoptotic response, highlighting differences in the mechanisms of regression for leiomyomas vs. other hormone-responsive tumor types. Additionally, this observation suggests that therapies that reduce estrogen availability to uterine leiomyomas do not result in apoptosis and helps explain the observed rapid regrowth of these tumors after cessation of currently available treatments.

	Materials and Methods

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Cell culture
ELT-3 uterine leiomyoma cells were maintained and propagated in culture on plastic (Corning, Corning, NY) in DF8 medium containing 10% FBS (HyClone Laboratories, Logan, UT) as previously described (26). For the purpose of studying the estrogen and tamoxifen responsiveness of ELT-3, cells were grown in estrogen-depleted medium containing phenol red-free DF8 and 10% charcoal-dextran-stripped FBS (DF8/PRF; HyClone). It was previously determined that tamoxifen had the greatest growth inhibitory effect on ELT-3 in 10% stripped serum (29). All studies were performed by plating cells in DF8 medium and allowing them to attach and grow for 72 h. Wells were then rinsed twice with sterile PBS, and the media were changed to the indicated conditions.

Growth kinetics were determined by plating 500 cells/well in triplicate on 24-well dishes and refeeding with fresh DF8, serum-free medium [DF8/PRF without serum or insulin plus 1% culture-grade BSA (Sigma Chemical Co., St. Louis, MO)], DF8/PRF, or DF8/PRF plus 5 µM tamoxifen. Tamoxifen was prepared from tamoxifen citrate salt (Sigma Chemical Co.) and stored in 70% ethanol at -20 C as 1 x 10^-5- and 5 x 10^-5-M stocks. Before use, stocks were diluted in medium at a 1:1000 ratio to obtain the appropriate final concentrations. At each indicated time point, cells were trypsinized and counted using a Coulter counter (Coulter Electronics, Hialeah, FL). One-way ANOVA followed by Fisher’s least significant difference (LSD) test of the log transformation of cell numbers after 96 h of exposure were used for statistical analysis.

Flow cytometry
Cells were plated at low density in T-150 flasks (Corning) and treated with the following: DF8 medium, serum-free medium, DF8/PRF, DF8/PRF plus 1 µM tamoxifen, and DF8/PRF plus 5 µM tamoxifen. After 48 h of treatment, 5-bromo-2'-deoxyuridine (BrdU; Sigma) was added to each flask at a final concentration of 10 µM, and cultures were incubated for 6 h at 37 C before harvest by trypsinization. Growth and wash media were collected and processed to include any cells that might have detached during treatment. After isolation and rinsing, cells were resuspended in 3 ml PBS, fixed by the addition of 1.5 ml cold 100% ethanol, and stored at -20 C overnight. Cells were permeabilized by incubation in 2.0 N HCl with 0.5% Triton X-100 (vol/vol; Sigma) at room temperature for 30 min. Cells were resuspended in 1 ml 0.1 M sodium borate, pH 8.5, to neutralize residual acid. A portion of the suspension was counted using a Coulter counter, and an aliquot of 1 x 10⁶ cells was incubated in a solution containing 50 µl 0.5% Tween-20 (vol/vol; Sigma) plus 1.0% BSA (wt/vol) in PBS and 20 µl fluorescein isothiocyanate (FITC)-labeled anti-BrdU (Becton Dickinson Immunocytometry Systems, San Jose, CA) for 30 min at room temperature. Cells were finally resuspended in 0.5 ml PBS containing 5 µg/ml propidium iodide (PI) and stored at 4 C in the dark until analyzed on a fluorescence-activated cell sorter (FACS).

Cell suspensions were analyzed using a Coulter EPICS Elite flow cytometer (Coulter) equipped with a 488-nm argon laser, and subsequent data analysis was performed on 1.0–2.0 x 10⁴ cells for each treatment using Coulter Elite software (Coulter). The lower limit for FITC fluorescence, and thus BrdU incorporation, was set based on control values for background fluorescence from previous experiments. Gating of events into G0/G1, S, and G2/M phases of the cell cycle was based on the PI fluorescence frequency histogram for ELT-3 cells grown in DF8 medium. Using these parameters and restrictions based on the forward scatter vs. PI fluorescence profile of serum-starved ELT-3 cells, the criteria for apoptosis were established. These limits remove debris and nonviable cells from analysis of the less than diploid (sub-2N) population.

Terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labeling (TUNEL assay)
Cells were plated on plastic chamber slides (Nunc, Naperville, IL) at a density of 500 cells/well, and the medium was changed to the following: DF8 medium, serum-free medium, DF8/PRF, DF8/PRF plus 1 µM tamoxifen, or DF8/PRF plus 5 µM tamoxifen. After 48 h, growth media were discarded, and the slides were processed using the In Situ Cell Death Detection Kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s protocol for cells grown in monolayer culture. Counterstaining was performed using a 0.05 µg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma) solution in PBS for 10 min before mounting in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) for fluorescence microscopy.

DNA ladders
ELT-3 cells were plated at low density in T-150 flasks and treated with the following: fresh DF8 medium, serum-free medium, DF8/PRF, DF8/PRF plus 1 µM tamoxifen, and DF8/PRF plus 5 µM tamoxifen. After 48 h of continuous treatment, cells were rinsed and trypsinized, collecting both growth and rinse media to ensure that detached cells were retained for analysis. Cells were lysed in 2–3 ml lysis buffer [100 mM NaCl, 50 mM Tris, 10 mM EDTA, 0.5% SDS (wt/vol), and 0.5 mg/ml proteinase K, pH 8.1] for 1 h at 37 C. Nucleic acids were then isolated by phenol-chloroform extraction and ethanol precipitation. The resulting pellet was resuspended in 1.5 ml TE buffer (10 mM Tris and 1 mM EDTA, pH 7.5). RNA was digested by adding 4 U/ml ribonuclease A and incubating for 1 h at 37 C. The resulting genomic DNA was precipitated as before, and the pellet was resuspended in TE buffer. Samples were electrophoresed for approximately 18 h on a 1% agarose (Life Technologies, Grand Island, NY) gel at 20 V. Gels were subsequently stained in a 5 µg/ml solution of ethidium bromide before photography.

Estrogen effects on cell growth in serum-free medium
Cells were plated in 24-well plates in triplicate at 500 cells/well for each growth condition. After 72 h, the cells were treated with either serum-free medium plus 0.1% ethanol or serum-free medium plus 10^-8 M 17ß-estradiol (Sigma), which was prepared as a 10^-5-M stock in 100% ethanol and stored at -20 C. Cells were counted every 24 h from 48–120 h as described above. The rate of cellular proliferation was determined by adding [³H]thymidine (Amersham Life Science, Arlington Heights, IL) to identical cultures 24 h before harvest. Nucleic acids were then isolated by lysing cells in a buffer of 1.0% SDS (wt/vol) and 0.3 N NaOH before trichloroacetic acid precipitation. Incorporation was determined by scintillation and expressed as counts per min/cell. Cell number after 120 h and [³H]thymidine incorporation were subjected to log transformation and Student’s unpaired t test.

To determine the rate of apoptosis under the above conditions, 2.5 x 10³ cells were plated into 60-mm² dishes. Each dish contained three 18-mm diameter sterile glass coverslips to which cells adhered and grew. The TUNEL assay was performed as previously described after 48 h of exposure. The numbers of both positive and negative figures for TUNEL labeling were counted by taking three passes per coverslip in a similar fashion at x250 magnification. Positive cells were expressed as a percentage of the total cells counted per coverslip. Statistical analysis of apoptotic rates was performed using Student’s unpaired t test.

Tamoxifen treatment in vivo
Intact 12-month-old female Eker rats were anesthetized, and 100-mg, 60-day release tamoxifen pellets were implanted above the shoulder sc. Implants were replaced after 60 days, and animals were killed after a total of 4 months of treatment. Age-matched untreated females were included as controls. At necropsy, uterine tumors were excised and fixed in 10% neutral-buffered formalin before paraffin embedding. Vaginal histology was used to determine the stage of estrus for each animal at the time of death. The care and handling of rats were according to NIH guidelines in Association for the Accreditation of Laboratory Animal Care-accredited facilities, and all protocols involving the use of these animals were approved by the Institutional Animal Care and Use Committee.

The TUNEL assay was performed using the kit listed above as indicated by the manufacturer for paraffin-embedded tissue sections. Hematoxylin- and eosin-stained adjacent sections were used to map each slide before quantitation to exclude any normal or necrotic tissue from consideration. The apoptotic rate of each tumor was then determined by counting the number of TUNEL-positive cells per 10 high power fields within regions of neoplastic tissue only. Results were analyzed using one-way ANOVA and Fisher’s LSD test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Growth arrest by serum starvation, estrogen withdrawal, and tamoxifen treatment
The rat uterine leiomyoma cell line ELT-3 is of confirmed smooth muscle origin, expresses functional estrogen receptors, and has been shown to proliferate in response to exogenous 17ß-estradiol in culture (26, 29). To determine the effects of estrogen withdrawal and tamoxifen on the growth of ELT-3, cells were grown under culture conditions that maximally or minimally stimulated cell growth [complete (DF8) and serum-free media, respectively] and in estrogen-depleted medium and estrogen-depleted medium containing 5 µM tamoxifen (Fig. 1). After 96 h of exposure, differences in cell number between all treatment groups and cells grown in complete medium were statistically significant (P < 0.01). Relative to logarithmically growing cells in complete medium, estrogen depletion reduced cell number by 45%. Treatment with 5 µM tamoxifen in estrogen-depleted medium resulted in an additional decrease in cell number, inhibiting ELT-3 growth to 86% of the control value. Serum starvation also arrested the growth of cells in culture and reduced cell number by 93% relative to that of cells grown in complete medium. Growth of cells in serum-free medium induced a portion of the cells to detach from the growth surface. Although this process did not occur in cultures treated with tamoxifen, cells did appear highly vacuolated within 24 h of tamoxifen exposure. Neither of these changes was witnessed in cultures grown in complete or estrogen-depleted medium.

View larger version (21K): [in this window] [in a new window]   Figure 1. Growth inhibition by estrogen withdrawal and tamoxifen treatment. ELT-3 cells were grown in 10% FBS, serum-free medium, 10% stripped serum, and 5 µM tamoxifen in 10% stripped serum. Cell numbers are plotted as a function of time ± SEM. Statistical analysis was performed by one-way ANOVA and Fisher’s LSD test on the log transformation of cell numbers. Estrogen-depleted, tamoxifen-containing, and serum-free media significantly reduced cell number over 96 h of exposure (P < 0.01).

The proliferative status and cell cycle distribution of ELT-3 cells grown in complete, serum-free, estrogen-depleted, and estrogen-depleted media containing 1 or 5 µM tamoxifen for 48 h were examined using FACS to measure cell proliferation based on BrdU incorporation and to determine the total DNA content of each cell using PI staining. Quantitation of BrdU incorporation into newly synthesized DNA indicated that media lacking estrogen or containing tamoxifen inhibited the proliferation of ELT-3 cells compared to that of cells grown in complete medium (Fig. 2A and Table 1). During the 6-h BrdU exposure, only 16% of cells grown in the absence of estrogen had undergone DNA replication. Tamoxifen also inhibited proliferation, reducing the number of labeled cells to 13% of the control value. In comparison, 82% of ELT-3 cells grown in complete medium and only 7% of those exposed to serum starvation had incorporated BrdU into newly synthesized DNA, indicating that estrogen withdrawal, tamoxifen treatment, and serum starvation were effective at arresting cell growth.

View larger version (31K): [in this window] [in a new window]   Figure 2. BrdU incorporation and DNA content. ELT-3 cells were grown in 10% FBS, serum-free medium, 10% stripped serum, 1 µM tamoxifen, and 5 µM tamoxifen in 10% stripped serum for 48 h. Before harvest, cultures were exposed to BrdU in growth medium for 6 h. Suspensions were labeled with both a FITC-conjugated anti-BrdU antibody and PI before flow cytometric analysis. A, Frequency histogram of BrdU incorporation by FITC fluorescence. B, Frequency histogram of DNA content by PI fluorescence. Estrogen withdrawal and tamoxifen treatment decreased cell proliferation without the induction of a large sub-2N apoptotic population that was observed in serum-free medium (indicated by arrow).

The TUNEL assay is used as a marker for DNA integrity in situ (30). The 3'-ends of damaged DNA are labeled by enzymatic incorporation of marker-conjugated free nucleotides in a template-independent manner. The biochemical preference of the terminal deoxynucleotide transferase enzyme for free DNA ends gives rise to the preferential labeling of apoptotic cells (31) and makes this assay a valuable tool in conjunction with other methods for the detection of programmed cell death.

Cultures of ELT-3 cells were grown and treated as described above for 48 h before examination by TUNEL. Cells grown in complete medium were negative for the presence of apoptotic cells. In agreement with FACS analysis, serum starvation produced the DNA fragmentation and micronuclei associated with apoptosis and detectable by TUNEL in 3.6% of cells (Fig. 3). Treating cells with estrogen-depleted medium or 5 µM tamoxifen produced low values for apoptotic cells labeled by the TUNEL assay of 0.1% and 0.2%, respectively.

View larger version (121K): [in this window] [in a new window]   Figure 3. TUNEL assay. ELT-3 cells were grown in 10% FBS (A), serum-free medium (B), 10% stripped serum (C), and 5 µM tamoxifen (D) in 10% stripped serum. DNA fragments were 3'-end labeled in situ with a FITC-conjugated deoxy-UTP and terminal deoxynucleotide transferase, with subsequent DAPI counterstaining. Apoptotic cells were not detected in 10% FBS, and the frequencies of TUNEL-positive cells in serum-free medium, 10% stripped serum, and 5 µM tamoxifen were 3.6%, 0.1%, and 0.2%, respectively.

View larger version (47K): [in this window] [in a new window]   Figure 4. DNA ladders. Genomic DNA isolated from ELT-3 cells grown in 10% FBS, serum-free medium, 10% stripped serum, 1 µM tamoxifen, and 5 µM tamoxifen in 10% stripped serum was electrophoresed on a 1% agarose gel. A distinct DNA ladder was present in cells grown in the absence of serum.

View larger version (16K): [in this window] [in a new window]   Figure 5. Estrogen rescue of serum starvation. ELT-3 cells grown in serum-free medium were exposed to either 10-8 M 17ß-estradiol (E2) or ethanol vehicle. Cell counts are plotted as a function of time ± SEM. Statistical analysis was performed by Student’s unpaired t test on the log transformation of cell numbers. Estrogen treatment significantly increased cell number after 96 h of exposure (P < 0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Flow cytometry indicated that estrogen withdrawal and tamoxifen treatment decreased the percentage of proliferating cells in each of these cultures. This was demonstrated by a decrease in both BrdU incorporation and the number of cells in S phase of the cell cycle. Additionally, the proportion of cells in G0/G1 increased with a concomitant decrease in G2/M. This occurrence indicated that treatment of cells with estrogen-depleted medium or tamoxifen induced a G1 block in the cell cycle that paralleled data obtained from estrogen deprivation of the human breast cancer lines MCF-7 and T-47D (32, 33). Under all treatment conditions that arrested cell growth, there remained a significant subpopulation of cells with a G2/M DNA content. Whether this is due to the presence of an additional G2 arrest or to the presence of tetraploid cells in the population is not clear. ELT-3 cells have a bimodal chromosome distribution (our unpublished results), thus cells in G1 with a 4N DNA content could contribute to the percentage of cells falling within parameters for G2/M.

When ELT-3 cells were serum starved, they produced DNA fragmentation that was detectable using the flow cytometer, the TUNEL assay, and genomic DNA electrophoresis. Quantitation of DNA fragmentation using these assays suggested that only very low to undetectable levels occurred in estrogen-depleted and tamoxifen-treated cultures after 48 h. In serum-starved cells, 3.6% and 25% of the cell population were determined to be apoptotic by TUNEL and FACS analysis, respectively. The TUNEL assay was used to quantitate apoptotic cells present in monolayer only and would not detect apoptotic cells that had detached from the dish. For this reason, the percentage of apoptotic cells detected with the TUNEL assay was less than that observed by FACS analysis in which detached cells in the medium were included in the analysis. The growth inhibition in tamoxifen-treated cultures (86%) was roughly equivalent to that observed during serum starvation (93%); however, the primary cause of growth arrest due to tamoxifen exposure, like that due to estrogen withdrawal, was inhibition of cell proliferation by blocking the exit of cells from the G1 phase of the cell cycle.

Interestingly, in ELT-3 cells, tamoxifen demonstrated an additional growth inhibitory effect (86% of control) over that obtained by estrogen depletion (45% of control) when it was used in an estrogen-free environment. This effect suggested that tamoxifen can inhibit cell growth by means other than competitive binding to the estrogen receptor. The use of tamoxifen as well as other antiestrogens in other in vitro tumor models has yielded similar results, sometimes regardless of the estrogen receptor status of the cells (29, 32, 34, 35, 36). These effects could result from binding of compounds to unique antiestrogen sites on tumor cells and/or blocking of the unliganded estrogen receptor’s ability to transduce growth signals from other pathways (37, 38, 39, 40). Although the mechanisms of growth suppression by tamoxifen remain unknown, apoptosis was not enhanced by this compound, indicating that inhibition of proliferation is the primary response of ELT-3 cells to this antiestrogen as well as to estrogen withdrawal.

In this study, treatment of leiomyomas with tamoxifen in situ did not cause a significant change in the apoptotic rate of neoplastic tissues. Previous reports from this laboratory have shown that tamoxifen clearly inhibits the growth and estrogen-stimulated expression of progesterone receptor messenger RNA in cells derived from Eker uterine leiomyomas (29, 41). Together, these data show that although tamoxifen can inhibit the growth of leiomyoma cells and is an estrogen antagonist in this tissue, it does not appear to cause the induction of apoptosis in vitro or in vivo.

The response of the human uterus to tamoxifen treatment appears to be complex and regulated at the level of the individual cell types within the organ. The incidence of endometrial carcinoma in separate therapeutic trials was increased in women receiving tamoxifen (42, 43, 44). Rapid growth of individual uterine leiomyomas in response to tamoxifen treatment has been reported; however, these incidents appear to be anecdotal, and no systematic study of the effect of tamoxifen on the myometrium has been undertaken. Tamoxifen is metabolized similarly in rats and humans and displays mixed agonist/antagonist activity in both species (45, 46). In mature intact rats, tamoxifen treatment reduces uterine wet weight and causes the disappearance of endometrial glands, consistent with antagonistic effects, but shows partial agonist activity with respect to the luminal epithelium similar to observations in the human uterus (27, 46). Tamoxifen has been shown to stimulate the transcriptional activation function-1 of the estrogen receptor, and this activity may be responsible for the partial agonist activity of this compound in some systems (47, 48). However, whether this activity appears to be capable of stimulating cell proliferation is species and tissue specific (27, 46). In addition to data in this and a previous report (29) in which tamoxifen inhibited myometrial cell growth in stripped serum, treatment of ELT-3 cells with 5 µM tamoxifen in serum-free medium inhibits cell proliferation by approximately 40% (data not shown). Therefore, tamoxifen does not appear to act as a partial agonist in this cell type and is unable to stimulate cell proliferation even under estrogen-deficient conditions.

The growth of the myometrium during periods of increased estrogen secretion, such as pregnancy, is primarily due to cellular hypertrophy, resulting in an increase in intracellular volume (49). Uterine leiomyoma growth is similarly stimulated by estrogen and affected by hormonal changes during the menstrual cycle (50); however, in fibroids, this hormone appears to stimulate cell proliferation as well. The treatment of leiomyomas with GnRH analogs reduces tumor volume by reducing cell size. This idea is supported by observations that cellularity, or the number of cells per given area of microscopic specimen, increases after treatment with GnRH agonists (51). Additionally, cell loss has not been observed in microscopic sections of GnRH-treated leiomyomas (22). Results from our current studies indicate that transformed myometrial cells are insensitive to the induction of apoptosis upon hormone deprivation and explain the rapid increase in tumor volume following the termination of therapy.

The inability of hypoestrogenism to induce cell death emphasizes the need for improved modalities of treatment for uterine leiomyomas. Tamoxifen belongs to a particular class of antiestrogens known as selective estrogen receptor modulators (41). These compounds display cell type specificity in their agonist/antagonist activity, and some have been shown to inhibit the effects of estrogen in the breast and endometrium while preventing bone loss and lowering serum cholesterol levels (52, 53). The ability of particular antiestrogens to induce a hypoestrogenic effect on fibroids without the side-effects that accompany treatment with GnRH analogs offers the possibility of treating women for extended periods of time without the need for surgery or hormone add-back. In addition, the fact that transformed myometrial cells appear to remain competent for the induction of apoptosis could be instrumental in the development of novel therapeutic techniques.

	Acknowledgments

 
The authors thank Dr. Tim McDonnell for his expert advice concerning apoptosis detection, Mr. Dennis Walker for his technical assistance and expertise in flow cytometric analysis, and Dr. Sandra Dunn and Dr. Darlene Dixon for critical review of this manuscript.

	Footnotes

 
¹ This work was supported in part by NIH Grants CA-72253 and HD-33605 (to C.W.) and CA-16672.

Received October 14, 1996.

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