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Regular Article TA |
a Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park Research Division, Smithville, Texas 78957
b Laboratory of Women's Health, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
ABSTRACT
Uterine leiomyomas are responsive to the ovarian steroids, estrogen and progesterone; however, a mechanistic understanding of the role of these hormones in the development of this common gynecologic lesion remains to be elucidated. We have used the Eker rat uterine leiomyoma model to investigate how ovarian hormones regulate or promote the growth of these tumors. Proliferative and apoptotic rates were quantitated in normal uterine tissues and leiomyomas in response to endogenous ovarian steroids. In 2- to 4-mo-old animals, cell proliferation in the normal uterus corresponded with high serum levels of steroid hormones during the estrous cycle, and apoptosis occurred in the rat uterus in all cell types following sharp, cyclical declines in serum hormone levels. It is interesting that the responsiveness of uterine mesenchymal cells changed between 4 and 6 mo of age, with significant decreases in both proliferative and apoptotic rates observed in myometrial and stromal cells of cycling animals. Leiomyomas displayed much higher levels of proliferation than did age-matched myometrium; however, their apoptotic index was significantly decreased in comparison with normal myometrium. This disregulation between proliferative and apoptotic responses, which were tightly regulated during ovarian cycling in the normal myometrium, may contribute to the disruption of tissue homeostasis and underlie neoplastic growth of these tumors.
aging, apoptosis, estradiol, female reproductive tract, progesterone, steroid hormones, uterus
INTRODUCTION
Ovarian production of the steroid hormones estrogen (E2) and progesterone (Pg) is responsible for the cyclical preparation of a uterus for implantation of a newly fertilized embryo. This process includes proliferation and differentiation of epithelial and stromal cells that constitute the uterine endometrium. Layers of smooth muscle, or myometrium, surround and support endometrial tissue and make up the bulk of uterine volume. Like the endometrium, myometrial cells possess receptors for both E2 and Pg, and changes in the circulating levels of these hormones regulate myometrial physiology [1, 2]. For example, the expression of autocrine- or paracrine-acting growth factors (or both) by the myometrium in response to E2 may be important for the signaling of uterine cells to replicate during the proliferative phase of the reproductive cycle [3–5]. E2 and Pg also play a role in regulating the contractile response of uterine smooth muscle during pregnancy [6].
Uterine leiomyomas, commonly referred to as fibroids, are benign tumors originating in the myometrium. These tumors are the most common neoplasm of the reproductive tract in premenopausal women, with an incidence that has been reported to be as high as 77% [7]. When fibroids are symptomatic, they are associated with dysmenorrhea and menorrhagia, and are also a common cause of infertility [8]. Because of their prevalence, symptomatic fibroids are the leading cause of hysterectomy in the United States and represent a significant health issue for women [9].
Uterine leiomyomas are responsive to ovarian hormones [10]. Data collected through clinical observation and therapeutic trials suggest that fibroid growth is regulated by E2, Pg, or both. The indication for hysterectomy due to symptomatic fibroids reaches a maximum incidence at about 45 yr of age and then declines precipitously, coinciding with the onset of menopause [9]. In addition, leiomyomas in women of reproductive age are commonly treated with hormone ablative therapies such as GnRH agonists [11]. Treatment with these agents results in the shutdown of ovarian hormone production by inhibiting the release of gonadotropins by the pituitary and the halting of follicular development in ovaries [12, 13].
The need exists to determine how hormones control tumor biology at a molecular level in order to develop more specific treatments for fibroids without limiting side effects. The lack of understanding of the hormone responsiveness of fibroids can be attributed in part to the need for a reliable in vivo model that recapitulates the human disease. Our laboratory has recently described an animal model, the Eker rat, in which uterine leiomyomas develop spontaneously in females that are heterozygous for a germline mutation of the tuberous sclerosis 2 (Tsc-2) tumor suppressor gene [14, 15]. These tumors are of smooth muscle origin as determined by their expression of desmin and smooth muscle actin. Eker leiomyomas, like human fibroids, are hormone-responsive. Ovariectomy of Eker females at 4 mo of age completely abolished the development of gross tumors in 16-mo-old animals, demonstrating the dependence of these tumors on ovarian hormones [16]. In vitro and in vivo growth of cells derived from Eker leiomyomas express receptors for both E2 and Pg, and are growth-stimulated by E2 [15]. The Eker model, therefore, appears to be an excellent model to acquire much needed information toward the understanding of the role of steroid hormones in the growth of uterine leiomyomas.
The purpose of the present study was to investigate how the growth of uterine leiomyomas is regulated by endogenous ovarian hormones and determine if proliferative responses, apoptotic responses, or both are aberrantly regulated in primary tumors in vivo. We first characterized the Eker model for ovarian hormone secretion and the response of the normal uterus to ovarian cycling. Serum concentrations of E2 and Pg and the proliferative and apoptotic rates of uterine tissues were determined at each day of the estrous cycle. The growth kinetics of leiomyomas were measured in aged females, compared with age-matched normal myometrium, and correlated with the hormone levels observed in those animals. We report that fundamental changes occur in the response of the myometrium to hormones throughout the lifetime of the animal as well as during tumorigenesis in this tissue. These changes ultimately alter tissue homeostasis by affecting the decision of myometrial cells to either divide or undergo apoptosis.
MATERIALS AND METHODS
Animals
Female rats from a closed colony on site were genotyped for carrier status of the Eker mutation in the Tsc-2 tumor suppressor gene, and maintained on a 14L:10D cycle with food and water provided ad libitum. Daily vaginal smears were performed to determine the regularity of reproductive cycling and were used to determine when animals would be killed on the basis of the estrous cycle. Noncarrier isogenic Eker females between the ages of 2–9 mo were used to establish the response of normal uterine tissues, and tumor-bearing carrier females between the ages of 12–16 mo were used for similar analyses of leiomyomas. Two h prior to killing, each animal received an i.p. injection of a sterile 20 mg/ml solution of 5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Company, St. Louis, MO) in PBS at a dose of 100 mg/kg. All animals were killed by CO2 asphyxiation between 1200 and 1600 h, at which time cardiac blood and tissue sections were harvested. The reproductive stage of each animal at the time of death was determined by serum hormone levels and vaginal and ovarian histology [17, 18]. The care and handling of rats were in accord with National Institutes of Health guidelines and Association for the Accreditation of Laboratory Animal Care-accredited facilities, and all protocols involving the use of these animals were approved by the M.D. Anderson Animal Care and Use Committee.
Serum Hormone Radioimmunoassays
Blood from study animals was allowed to clot overnight at 4°C, and cells were then precipitated from serum by centrifugation at 2000 x g for 10 min in a tabletop centrifuge. Serum samples were then stored at -20°C until processed. Serum 17ß-estradiol and Pg levels were determined by radioimmunoassay (RIA) using the Ultrasensitive Estradiol and Progesterone Coated Tube kits from Diagnostic Systems Laboratories, Inc. (Webster, TX) according to the manufacturer's protocols. Coefficients of variation (c.v.; inter-assay and intra-assay) were empirically determined for E2 and Pg measurements and determined to be in line with values supplied by the manufacturer. Inter-assay c.v. for E2 using the 20 pg/ml sample (n = 9) was 11.8%, and for Pg, using a 10 ng/ml sample (n = 8) was 12.9%; the manufacturer's inter-assay c.v. was reported as 9.7% and 12.9%, respectively. Intra-assay c.v. reported by the manufacturer was 6.5% for E2 (mean 
25 pg/ml) and 6.9% for Pg (mean 5 ng/ml), which was confirmed with duplicate samples in each assay and stabilized by averaging values from multiple assays, which yielded a computed c.v. of 8.0% and 9.1% for E2 and Pg (n = 9 and 8, respectively). Comparisons of serum hormone concentrations between phases of the estrous cycle were performed using one-way ANOVA and Fishers least significant difference (LSD) test.
BrdU Immunostaining
All incubations were performed at room temperature unless noted otherwise, and slides were washed between steps with a 0.1% solution of BSA in PBS. Tissue sections were deparaffinized and endogenous peroxidase activity was quenched in a solution of 1% H2O2 in methanol for 20 min. Double-stranded DNA was denatured by incubating slides in 1 N HCl at 40°C for 20 min. Slides were then treated with a 0.05% solution of Protease Type XXVII (Sigma) in deionized H2O for 20 min. The anti-BrdU antibody (Becton Dickinson, Lincoln Park, NJ) was diluted 1:25 in a solution of 0.1% BSA/0.5% Tween in PBS and applied to sections for 1 h. The secondary, biotinylated anti-mouse immunoglobulin G antibody (Vector Laboratories, Inc., Burlingame, CA) was diluted to 22.5 µl into 5 ml 0.1% BSA/PBS solution and allowed to incubate with sections for 30 min. Following the final incubation, BrdU-positive nuclei were visualized using the Vectastain ABC Elite Peroxidase Kit (Vector).
The proliferative rates of tissues were assessed in the following manner. BrdU-positive epithelial cells were counted in a section and expressed as a percent of the total number of epithelial cells. Positive-stained cells of the myometrium, stroma, and leiomyomas were determined as the average per high-powered field (HPF, 400x) of at least four fields for myometrium and stroma and 10 fields for leiomyomas. These averages were then normalized using the average number of total nuclei in a 1000x field for a particular group to account for differences in cellularity between cycle stage, animal age, and tissues. Statistical analyses were performed using student's one-sided t-test to examine tissue differences between age groups within a particular phase of the estrous cycle, and ANOVA and Fisher's LSD tests were used for tissue differences between various reproductive phases.
Leiomyosarcomas have been infrequently observed in this rodent model. Because the biology of sarcomas is quite different from benign fibroids, the analysis of tumors in this study was limited to leiomyomas only. One of the primary criteria for the diagnosis of leiomyosarcomas in women is based on the mitotic rate of the tumor [19]. To exclude sarcomas from consideration, we set a limit on BrdU incorporation at three standard deviations (SDs) from the mean of all leiomyoma samples examined. Using this criterion 1 tumor out of the 30 analyzed, with a rate of BrdU incorporation greater than four SD from the mean, was eliminated.
TUNEL Assay
Formalin-fixed uterine and leiomyoma sections were processed using the In Situ Cell Death Detection, Fluorescein kit from Boehringer-Mannheim Corporation (Indianapolis, IN). The manufacturer's protocol was followed for paraffin-embedded sections with the exception of a 90-min reaction incubation at 37°C. Quantitation of cell death rates was determined as described for BrdU immunostaining using fluorescent microscopy. Because of the difficulty in recognizing areas of gross necrosis and infarction under fluorescent conditions, leiomyoma sections were mapped using adjacent sections stained with hematoxylin and eosin to exclude such areas from consideration.
RESULTS
Cell Proliferation and Death in the Uterus
To study the mechanism by which ovarian hormones regulate leiomyoma growth, we began by characterizing the reproductive cycle of 2- to 4-mo-old (peripubertal) females. This was done to ensure that ovarian hormone secretion and uterine responses in Eker rats were normal relative to data reported in the literature and to provide baseline data for myometrial responses to ovarian cycling in this model. The estrous cycle of regularly cycling rats consists of 4–5 days and can be divided by ovulation, which occurs on the evening of proestrus. In peripubertal Eker females, E2 levels were highest during proestrus (P 
0.05), the time in which follicular growth and development occurs prior to ovulation (Fig. l). Following ovulation E2 levels declined sharply from 20.2 pg/ml in proestrus to 10.6 pg/ml in estrus and remained low for the remainder of the cycle. The Pg concentration in serum rose following ovulation from 25.3 ng/ml in estrus to 33.3 ng/ml in metestrus with the development of corpora lutea in the ovary.
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The proliferative rates of tissues were determined by visualizing BrdU incorporation into newly synthesized DNA by immunohistochemistry in tissue sections. Cell proliferation was first assessed in peripubertal uterine tissues as a function of the estrous cycle (Figs. 2 and 3). The myometrium and stroma displayed a peak of DNA synthesis during proestrus, coinciding with high levels of E2 secretion during this period by the ovaries. Myometrial cells showed a single wave of proliferation rising from 0.9 BrdU-positive nuclei/HPF in diestrus to 8.6/HPF in proestrus and returning to 3.8/HPF in estrus. This peak in myometrial proliferation was statistically different from all other phases of the cycle (P 0.05). Incorporation of BrdU into stromal DNA began to rise during diestrus and reached its greatest level of 22.7 BrdU-positive nuclei/HPF in proestrus before declining to 5.7/HPF in estrus. BrdU-positive stromal cells tended to lie just below the luminal epithelium in diestrus, but were more diffuse later in proestrus. Both epithelial cell types in the uterus underwent peak levels of DNA replication on the day of metestrus. In the luminal epithelium, 43.2% of cells incorporated BrdU during metestrus, and 34.2% of glandular epithelial cells were positive for BrdU staining.
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In proestrus, a qualitative difference was noted between uterine sections regarding BrdU incorporation in the luminal epithelium (Fig. 2A and 2B). Three of the five animals showed no incorporation of BrdU in the lumen. The vaginal sections from these animals displayed incomplete epithelial cornification (Fig. 3, inset); therefore, these samples were labeled early proestrus (EP). The two remaining animals had BrdU incorporation rates of 16% and 35% and showed complete vaginal cornification (late proestrus; LP). When females in proestrus were divided on the basis of these two patterns of uterine BrdU staining, a second peak of DNA synthesis in luminal epithelial cells became evident during LP, coinciding with high E2 levels. In addition, a statistically higher rate of proliferation was also observed in the myometrium during LP (14.9 BrdU-positive nuclei/HPF) versus EP (4.5/HPF; P 0.05). Both of these events correlated with a reduction in serum Pg concentrations from EP to LP (P 0.05).
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The rate of apoptosis of uterine cells in situ was determined by the TUNEL assay. Figure 4 shows that positive figures were identified in the peripubertal uterus by TUNEL and that the frequency differed with the stage of the estrous cycle. Cell death was maximal in each of the four uterine cell types on the day of estrus (Fig. 5). The rise in the apoptotic rate of myometrial cells from 5.7 TUNEL-positive nuclei/HPF in proestrus to 12.2/HPF in estrus represented a significant change (P 0.05) and correlated with the statistically lower serum E2 levels on that day. Apoptotic figures in the stroma were common throughout the estrous cycle, reaching a peak of 49.3 TUNEL-positive nuclei/HPF on the day of estrus. Contrary to the tendency of cells undergoing DNA synthesis to be located near the lumen, apoptotic cells in the stroma with positive stains were most prevalent in the basal area, next to the myometrium. Luminal and glandular epithelia reached rates of 8.7% and 6.0%, respectively. Visible micronuclei, the small membrane-bound figures resulting from the nuclear fragmentation that occurs during apoptosis, were present in the cells of TUNEL-stained uterine sections (Fig. 4).
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Myometrial Responses to Hormones Decrease with Age
The growth kinetics of uterine tissues in 6- to 9-mo-old (mature) females that were cycling normally were also determined. The timing of proliferation and apoptosis during the estrous cycle of mature uterine cells was similar to that of peripubertal animals (data not shown). However, we observed that BrdU staining of myometrial cells in mature animals was consistently less frequent than that of sections from the same estral stage in peripubertal animals. When the proliferative rates of mature uterine cells were compared with those of peripubertal females, a decrease was seen that was specific for cells of mesenchymal origin only; namely, the stroma and myometrium. Table 1 shows the results of BrdU staining in proestrus when cells of the myometrium and stroma proliferated at their greatest rate. Myometrial cells of peripubertal females showed a proliferative rate of 8.6 BrdU-positive nuclei/HPF compared with 3.6/HPF in mature females (P 0.05). If proestrus was restricted in both age groups to late proestrus when the 2- to 4-mo-old myometrium was previously observed to proliferate at its greatest rate, the difference between peripubertal and mature animals became even greater (14.9/HPF vs. 3.3/HPF; P 0.05). In the stroma, peripubertal and mature animals had values of 22.7/HPF and 6.8/HPF, respectively, for BrdU incorporation (P 0.05).
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Proliferative rates of cells of the epithelia did not decrease with age. In the lumen during proestrus, 10.2% of peripubertal epithelial cells were positively stained for BrdU compared with 9.2% of mature cells. No differences were noted between age groups during the peak of cell proliferation in metestrus nor when the comparison was restricted to animals in late proestrus only (data not shown). In addition, the proliferative rates of glandular cells in metestrus were 25% and 21% for peripubertal and mature cells, respectively.
TUNEL staining of uterine sections yielded similar results. Cell death rates during estrus, the time at which this process was observed to be maximal, decreased in the mesenchymal cells of older animals, which was commensurate with the observed decline in proliferation (Table 2). In the myometrium, the number of positive TUNEL figures decreased from 12.2/HPF in peripubertal females to 3.8/HPF in mature animals (P 0.05). Cells of the stroma underwent cell death at a rate of 49.3/HPF in the younger rats, whereas those of older animals showed a rate of only 20.6/HPF (P 0.05). The percentage of epithelial cells undergoing cell death during estrus declined slightly, but because of the variability observed, statistical differences were not noted. Therefore, although proliferation was decreased in the mesenchymal cells of the mature uterus, the rate of cell death was also decreased, thereby maintaining the balance of cell loss and replacement that is necessary for tissue homeostasis.
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Leiomyomas Possess a Defect in the Apoptotic Pathway
The incidence of uterine leiomyomas in the Eker model is approximately 30% in 12-mo-old females and increases to near 70% by 16 mo of age. Between these ages, prolonged periods were encountered in which the vaginal cytology of Eker females remained fixed, suggesting that ovarian hormone levels were static and no longer fluctuated normally (data not shown). The Eker mutation exists on the background of the Long-Evans strain of rats. The ovarian histology and associated E2 and Pg levels for aged rats of this strain have been previously documented [17, 20]. Based on our data and that available from the literature, aged Eker females were separated into three stages of reproductive senescence using vaginal and ovarian histology and serum hormone levels as criteria (Table 3). Pseudopregnant (PP) animals had ovaries with numerous hypertrophied corpora lutea and a mucified vaginal epithelium. Persistent estrus (PE) was characterized by follicular cysts in the ovaries and a thickened, often cornified vaginal epithelium. Females were labeled atrophic (AT) based on the absence of follicular structures in the ovaries and the unstimulated appearance of the vaginal epithelium. E2 levels did not vary greatly between groups; however, large differences in Pg levels were seen, with PP animals having the highest (48.4 ng/ml) and AT animals the lowest (10.2 ng/ml).
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Although we observed that the rates of cell replication and death changed in normal myometrium and stroma with age, these processes both declined, leaving the homeostatic balance in place. Uterine leiomyomas assessed in 12- to 16-mo-old Eker rats showed greatly increased rates of proliferation relative to the aged myometrium (Fig. 6). In PP, PE, and AT animals, the rates of BrdU incorporation in tumors were 24-fold greater than those of the corresponding normal tissue (P 0.05). A trend toward increased proliferation was present in PE leiomyomas (13.1 BrdU-positive nuclei/HPF) relative to PP (9.1/HPF; P = 0.17) and AT (7.4/HPF; P = 0.06) tumors, although these differences were not statistically significant. No difference in tumor volume existed between the three stages of reproductive senescence (data not shown).
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Quantitation of TUNEL staining revealed that an overall statistically lower rate of apoptosis existed in leiomyomas compared with normal myometrium (P 0.05; data not shown). However, when animals were separated on the basis of stage of reproductive senescence, it was observed that the difference in apoptotic rates was specific for PE animals only. In this group, the apoptotic rate of the normal myometrium was 2.4 TUNEL-positive nuclei/HPF and was at least eightfold greater than the rates seen in myometrial cells of either PP or AT females (P 0.05; Fig. 7). Leiomyomas, on the other hand, showed no difference in apoptotic rates under the three different hormonal milieus and did not exhibit an elevated rate of cell death in PE, as was seen in normal tissue. The apoptotic rate of the myometrium was more than fourfold higher than leiomyomas in this group (P 0.05). From these observations it appeared that leiomyomas not only had a much greater rate of proliferation, but also lost the ability to undergo apoptosis at a time when the normal myometrium did. Consequently, in tumors, defects that resulted in increased proliferation and decreased apoptosis led to a disruption in the balance of cell loss and renewal relative to the normal tissue.
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DISCUSSION
This study investigated the mechanism by which endogenous ovarian hormones regulate the growth of myometrium and uterine leiomyomas in the Eker rat model. Serum concentrations of E2 and Pg were measured, and the proliferative and apoptotic rates of tissues were determined for animals at various reproductive stages. It was found that the normal myometrium of peripubertal animals proliferated at its greatest rate on the day of proestrus, the time at which serum E2 concentration peaked. Apoptosis occurred in all cell types of the uterus and was maximal when E2 and Pg levels were both low. With increasing age, decreases in the rates of proliferation and apoptosis were observed in uterine cells of mesenchymal but not epithelial origin, suggesting that changes had occurred in the ability of these cells to respond to steroid hormones. Tissue homeostasis was maintained, however, because both the rates of cell renewal and loss were diminished. In contrast, leiomyomas showed increased rates of proliferation, suggesting that they had a reduced requirement for steroid hormones for continued cell proliferation. In addition, these tumors appeared to have lost the ability to appropriately regulate apoptosis, thereby disrupting the balance required for normal tissue homeostasis.
The maximal rate of myometrial BrdU labeling at the time the concentration of E2 in serum was greatest suggests that cell division in this tissue is driven by E2. Studies reporting the mitotic response of myometrial cells to hormonal manipulation are rare, apparently because the appropriate stimuli needed to induce DNA synthesis in this tissue are complex and remain elusive. Pg alters the proliferative effects of E2 in uterine tissues, and stromal proliferation has been shown to require Pg priming prior to E2 exposure [21, 22]. Myometrial cells, however, were not stimulated to divide with this hormonal regimen. In contrast, the correlation between decreased Pg secretion and increased myometrial BrdU incorporation in 2- to 4-mo-old, late proestrus animals suggests that Pg could negatively regulate DNA synthesis in these cells. Uterine distention has been shown to stimulate the proliferation of the myometrium [23]. However, the increase in cellular hypertrophy and uterine water inhibition in response to E2 during the estrous cycle are unlikely to have profound effects on myometrial cell number. This indirect effect of E2 on myometrial mitosis would likely be seen in ovariectomized rodents in which uterine volume increases severalfold with E2 treatment [21].
Apoptosis was seen in all uterine cell types of peripubertal animals in this study, including the myometrium. The maximum rate of cell death occurred coordinately in each of these cell types when both E2 and Pg serum concentrations were low, suggesting that one or both of these hormones inhibits apoptosis in the uterus. Previous in vitro studies have suggested that cell lines derived from Eker rat uterine leiomyomas have an apoptotic defect. Growth in medium devoid of steroid hormones is greatly reduced relative to serum-containing medium, but kinetic studies indicate that decreased cell numbers are the result of decreased cell proliferation, with no significant increase in apoptosis [24]. These same cells can, however, be induced to undergo apoptosis by serum-starvation, suggesting that the apoptotic program in these cells is intact, but is refractory to induction by hormone depletion. However, previous studies investigating the role of E2 and Pg on the regulation of apoptosis in the uterine epithelium suggest that, although withdrawal of both E2 and Pg probably participates in the induction of apoptosis in uterine epithelium, Pg plays a stronger role in modulating the rate of cell death in this uterine compartment [25, 26].
Our data indicate the ability of E2 to stimulate cell division in the myometrium is age-dependent. It has been previously reported that the administration of E2 to sexually immature rats (20 days) stimulates mitosis in the myometrium [27], whereas this response is minimal in ovariectomized, 3- to 4-mo-old rodents [21]. The decreased rates of proliferation and apoptosis observed in myometrium and stroma of 6- to 9-mo-old animals in our study suggests that responsiveness to changes in ovarian hormone levels is dampened in these tissues with increasing age. Data suggest that the ability of E2 to stimulate DNA synthesis in myometrium and stroma may be impaired in older animals because of changes in both receptor number and function [28, 29].
Apoptosis is important for the proper maintenance of homeostasis in a tissue and the removal of damaged or excess cells from a population. Ovarian hormones induce proliferation of uterine cells during each reproductive cycle, and this process has been shown to occur in the human and rodent uterine endometrium as a means of maintaining cell number in this tissue [30, 31]. We have previously demonstrated by DNA laddering, flow cytometry, and TUNEL that apoptosis occurs in Eker leiomyoma cell lines in response to serum starvation, but not in response to depletion of steroid hormones from the growth medium [24]. In the present experiments, TUNEL staining demonstrated that apoptosis occurred in the uterus of Eker females; however, the decreased rate of apoptosis in leiomyomas relative to age-matched myometrium indicated that a defect in the regulation of this process exists in these tumors. A study by Matsuo et al. [32], which investigated the expression of the Bcl-2 protein in human fibroid tissue by Western blotting, showed that this apoptotic inhibitor was abundantly present in tumors, but undetectable in adjacent myometrium. This observation may provide a mechanism for the reduced rates of cell death observed in Eker tumors and suggests that defects in the regulation of apoptosis may contribute to the growth of human leiomyomas as well.
We observed that proliferation in leiomyomas occurred at relatively low levels of E2, suggesting an increased sensitivity of these tumors to steroid hormones. In addition to the observation that ovariectomy of Eker females at a young age prevented tumor development, we have shown that treatment of Eker rats with the antiestrogens, tamoxifen or raloxifene analogue LY 326315, inhibit both tumor incidence and cell proliferation [16]. Therefore, signaling through the ER is important in the growth of Eker leiomyomas, suggesting that these tumors may be hypersensitive to the proliferative effects of estrogens and require much lower levels of E2 than those necessary to stimulate DNA synthesis in the normal tissue of young animals.
The ability to shrink fibroid volume in premenopausal women by inhibiting ovarian function is limited because of the adverse effects of a hypoestrogenic milieu on other organ systems. GnRH agonist treatment in premenopausal women is associated with accelerated bone resorption and an increase in serum cholesterol levels [33–35]. Because of these facts, the duration of GnRH therapy is limited, and symptomatic fibroids are ultimately treated surgically by myomectomy or hysterectomy. The heavy reliance on surgical intervention for the treatment of symptomatic fibroids, which results in more than 200 000 hysterectomies annually is unacceptable, and the need for improved modalities of therapy is apparent. Dissection of the factors that contribute to tumor development and growth will facilitate the discovery and implementation of better treatments. We have shown that Eker leiomyomas have defects in their apoptotic responses as well as increased cellular proliferation. In the normal myometrium, cell replication and death were tightly regulated by ovarian cycling; however, hormonal regulation of these processes in tumors was altered. An increased sensitivity of leiomyoma cells to steroid hormones may underlie the observed disruption of normal growth kinetics. If this is the case, tissue-specific antihormones could allow treatment of women for extended periods of time without the detrimental effects of hypoestrogenism. In addition, agents that have the ability to stimulate the apoptotic rate of fibroids could improve both current and future medicinal therapies.
ACKNOWLEDGMENTS
We thank Dr. Claudio Conti and Dr. Sandra Dunn for their critical review of the manuscript.
FOOTNOTES
First decision: 18 February 2000.
1 Supported in part by grants CA72253, ES08263, and CA16672 from the National Institutes of Health; and grant ES07784 from the National Institute of Health Sciences. 
2 Correspondence: Cheryl L. Walker, Department of Carcinogenesis, U.T.M.D. Anderson Cancer Center Science Park Research Division, Park Road 1C, Smithville, TX 78957. FAX: 512 237 2475; cwalker{at}odin.mdacc.tmc.edu
Accepted: June 7, 2000.
Received: January 18, 2000.
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 C. L. Walker Role of Hormonal and Reproductive Factors in the Etiology and Treatment of Uterine Leiomyoma Recent Prog. Horm. Res., January 1, 2002; 57(1): 277 - 294. [Abstract] [Full Text] [PDF]
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 K. D. Houston, D. S. Hunter, L. C. Hodges, and C. L. Walker Uterine Leiomyomas: Mechanisms of Tumorigenesis Toxicol Pathol, January 1, 2001; 29(1): 100 - 104. [Abstract] [PDF]
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