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Effects of progesterone on uterine leiomyoma growth and apoptosis
Steroids 65 (2000) 585–592
Effects of progesterone on uterine leiomyoma growth and apoptosis Takeshi Maruoa,*, Hiroya Matsuoa, Takashi Samotoa, Yosuke Shimomuraa, Osamu Kurachia, Zhijian Gaoa, Yin Wanga, Irving M. Spitzb, Elof Johanssonb a
Department of Obstetrics and Gynecology, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Center for Biomedical Research, The Population Council, New York, NY 10021, USA
Abstract Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. Recently we have found that the use of levonorgestrel-releasing intrauterine system (IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. These clinical experiences prompted us to characterize the effects of progestin on the proliferation and apoptosis of leiomyoma cells cultured in vitro. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either E2 (10 ng/ml) or P4 (100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells; whereas in cultures of normal myometrial cells, the addition of E2 augmented PCNA expression in the cells, but P4 did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P4 treatment resulted in an increase in EGF expression in the cells. In contrast, E2 treatment augmented EGF-R expression in cultured leiomyoma cells, but P4 did not. These results indicate that P4 up-regulates the expression of PCNA and EGF in leiomyoma cells, whereas E2 up-regulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium, suggesting that the abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Furthermore, Bcl-2 protein expression in leiomyoma cells was up-regulated by P4, but down-regulated by E2. Therefore, it seems likely that P4 may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Levonorgestrel-releasing IUS; Progesterone; 17-Estradiol; Uterine myoma; EGF; Bcl-2 protein
1. Introduction Uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium, occurring in as many as 30% of women over 35 years of age [1]. The majority of patients have multiple leiomyomas, and each leiomyoma is thought to be clonal, arising independently from a single smooth muscle cell [2]. Although the nature of the initial event is unknown, a role for ovarian steroid hormones in the growth of uterine leiomyomas is likely, because these tumors grow during the reproductive years, increase in size during pregnancy, and regress after menopause [3,4]. Fur* Corresponding author. Tel.: ⫹81-78-382-6000; fax: ⫹81-78-3826019. E-mail address: [email protected] (T. Maruo).
thermore, treatment with GnRH analogs, which reduces ovarian steroid hormone concentrations, leads to a reduction in the size of leiomyomas; however, enlargement of leiomyomas recurs after therapy with GnRH analogs is discontinued [5]. These findings suggest that leiomyoma growth is dependent on ovarian steroids. On the other hand, a growing body of evidence suggests that the action of estrogen may be mediated in part by local growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I), produced by the target cells [6 – 8]. The mechanisms of action of ovarian steroids in the regulation of leiomyoma growth, however, are not well defined as yet. Homeostatic control of the net growth of tumors is thought to be the result of the dynamic balance between cell proliferation and cell death; too much growth can come
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Fig. l. Hemoglobin levels in blood before and after insertion of LNg-IUS in menorrhagic women with intramural myomas. Bars represent mean ⫾ SD.
from too little death as well as from too much proliferation [9]. Only several years ago, many researchers trying to understand what causes the growth of tumors focused their attention on the pathways within cells that tell them when to divide. However, recent work is showing that cells also have internal pathways that tell them when to die [10]. It is possible that in tumors the death pathway may be suppressed, extending the lives of the cells. Actually, apoptosis, or programmed cell death, is known to occur in tumors either spontaneously or in response to treatment [11,12]. Recently we have found that the use of levonorgestrelreleasing intrauterine system (IUS) (LNg-IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas, because of a striking reduction in menorrhagia. Although some women with large intramural myomas had spontaneous expulsion of LNg-IUS at various intervals, they wanted reinsertion of the device because of remarkable reduction in menorrhagia. Significant increases in hemoglobin levels in blood were obtained after insertion of the devices (Fig. 1). No significant differences were noted in myoma volume and uterine volume, as assessed by MRI examination between pretreatment and 12 months of use. LNg-IUS was proven to be an effective modality for the long-term management of menorrhagia due to uterine myoma and adenomyosis. These clinical experiences prompted us to characterize the effects of progesterone on the proliferation and apoptosis of leiomyoma cells. Thus, we first determined the proliferative activity of leiomyoma cells compared with that of adjacent normal myometrial cells throughout the menstrual cycle by immunohistochemical analysis with a monoclonal antibody to proliferating cell nuclear antigen (PCNA) [13,14]. Furthermore, to understand the role of progesterone in regulating proliferative activity of leiomyoma cells, we examined whether ovarian steroids could influence PCNA expression in leiomyoma cells cultured under serum-free, phenol redfree conditions on the basis of immunocytochemical and immunoblot analyses. Because EGF plays a crucial role as a local factor in regulating leiomyoma growth [15–18],
possible effects of ovarian steroids on the expression of EGF and EGF-R receptor (EGF-R) in the cultured leiomyoma cells also were investigated. On the other hand, recent research efforts have focused on the function of proto-oncogene and tumor suppressor gene products in directing cell fate. In particular, an explosion of research interest has centered around the role of Bcl-2 in controlling the survival and death of cells. It is now evident that the Bcl-2 proto-oncogene encodes a 26-kDa protein that prevents the normal course of apoptotic cell death in a variety of cells induced by tropic factor deprivation or other stimuli without altering proliferation [19 –21]. It seems, therefore, that Bcl-2 protein may play an important role in the growth of tumors. However, to date, no information is available on the expression of Bcl-2 protein in uterine leiomyomas. Thus, we also determined the expression of Bcl-2 protein in leiomyomas in comparison with that in the normal myometrium by means of immunohistochemical techniques and immunoblot analysis with a monoclonal antibody to human Bcl-2 protein. Furthermore, to understand the role of progesterone in regulating the expression of Bcl-2 protein in uterine leiomyomas, we examined whether progesterone could influence the levels of Bcl-2 protein expression in leiomyoma cells cultured in vitro.
2. Proliferative potential of uterine myometria and leiomyomas throughout the menstrual cycle Immunohistochemical examinations of leiomyoma tissues and the adjacent normal myometrial tissues in the proliferative phase of the menstrual cycle demonstrated that PCNA label was positive only in a few normal myometrial cells (Fig. 2A), whereas it was present in a somewhat greater number of leiomyoma cells (Fig. 2B). Immunohistochemical staining for PCNA in those tissues in the secretory phase showed that only a few normal myometrial cell nuclei were positive for PCNA label (Fig. 2C), whereas
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Fig. 2. Immunohistochemical staining of PCNA in sections of normal myometrium (A) and leiomyoma (B) in the proliferative phase, and normal myometrium (C) and leiomyoma (D) in the secretory phase of the menstrual cycle. Bars ⫽ 5 m. Original magnification, ⫻400.
large numbers of the leiomyoma cell nuclei were positive for PCNA label (Fig. 2). Determination of the mean percentage of PCNA-positive nuclei in leiomyoma and the adjacent normal myometrial tissue sections revealed that the PCNA-positive rate was higher in leiomyoma cells than in normal myometrial cells throughout the menstrual cycle (P ⬍ 0.01) and that the PCNA-positive rate of leiomyoma cells was higher in the secretory, progesterone (P4)-dominated, phase than in the proliferative phase (P ⬍ 0.01). There were, however, no differences in PCNA-positive rate in normal myometrial cells in the proliferative phase and in the secretory phase (Fig. 3). The higher PCNA-labeling index in leiomyoma tissues, relative to the adjacent normal myometrial tissues throughout the menstrual cycle, may permit the enhanced growth of leiomyomas over the adjacent normal myometrium in the same uterus. With respect to the participation of P4 in the proliferation of leiomyoma cells, Kawaguchi et al. [22,23] reported that mitotic count in uterine leiomyomas is higher in the secretory phase of the menstrual cycle than in the proliferative phase and suggested that the growth of leiomyoma cells might be dependent not only on the presence of estrogen, but also on P4.
growth has been established [24], estrogen has received much attention as the major factor responsible for leiomyoma development. The mechanism underlying the stimulatory effects of progesterone on leiomyoma growth, however, has not been defined. To investigate the mitogenic effects of progesterone on uterine myometria and leiomyomas, an in vitro culture system of normal myometria and leiomyoma cells was established. Collagenase treatment of normal myometria and leiomyoma tissues provided a pure population of isolated cells with smooth muscle cell char-
3. Progesterone in the regulation of the proliferative potential of uterine myometria and leiomyoma cells Because leiomyoma growth is closely associated with reproductive years, and the vital role of estrogen in uterine
Fig. 3. The mean percentage of PCNA-positive nuclei in leiomyomas and the adjacent normal myometria as assessed by immunohistochemical analysis. Pro, Proliferative phase; Sec, secretory phase. *, P ⬍ 0.01.
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Fig. 4. Effects of E2 and P4 on PCNA protein expression in cultured normal myometrial cells and leiomyoma cells as assessed by Western immunoblot analysis. The 36-kDa PCNA protein was overexpressed in leiomyoma cells compared to normal myometrial cells. In normal myometrial cells, E2 increased the 36-kDa PCNA protein expression, but P4 did not. In leiomyoma cells, not only E2 but also P4 increased the PCNA protein expression in the cells.
acteristics without either stromal or glandular epithelial cell contamination. Immunohistochemical examination of the cultured cells revealed that these cells were immunostained with a monoclonal antibody directed toward the musclespecific protein desmin, but not immunostained with antibodies to either cytokeratin 19, a cytoskeletal protein specific for epithelial cells, or vimentin, a class of intermediate filament protein present in fibroblasts. Western immunoblot analysis showed that both cultured normal myometrial cells and leiomyoma cells contained immunoreactive PCNA with a molecular mass of approximately 36-kDa, and the 36-kDa PCNA expression in leiomyoma cells was more abundant than that in normal myometrial cells in untreated control cultures (Fig. 4). In cultures of normal myometrial cells, the addition of 17estradiol (E2, 10 ng/ml) remarkably increased the 36-kDa PCNA expression in the cells compared to that in control cultures, whereas such a remarkable increase in PCNA expression was not noted with the addition of P4 (100 ng/ml). By contrast, in cultures of leiomyoma cells, treatment with either E2 (10 ng/ml) or P4 (100 ng/ml) increased the 36-kDa PCNA expression in the cells relative to that in control cultures (Fig. 4). Furthermore, Western immunoblot analysis with a monoclonal antibody to EGF revealed that the cultured leiomyoma cells contained immunoreactive EGF with a molecular mass of approximately 133-kDa; the addition of P4 (100 ng/ml) to the serum-free medium resulted in an obvious increase in the level of expression of 133-kDa immunoreactive EGF, together with the appearance of immunoreactive EGF with a molecular mass of approximately 71-kDa, in the cells compared to that in control cultures (Fig. 5). By contrast, the addition of E2 (10 ng/ml) resulted
Fig. 5. Effects of E2 and P4 on EGF-like protein expression in cultured leiomyoma cells as assessed by Western immunoblot analysis. The addition of P4 (100 ng/ml) resulted in a remarkable increase in 133-kDa immunoreactive EGF expression, together with the appearance of 71-kDa immunoreactive EGF; whereas the addition of E2 (10 ng/ml) resulted in a somewhat lower expression of 133-kDa immunoreactive EGF relative to control cultures.
in a somewhat lower expression of 133-kDa immunoreactive EGF in the cells relative to that in control cultures. Instead, EGF-R expression in the cultured leiomyoma cells assessed by immunocytochemical analysis with a monoclonal antibody to EGF-R was notably augmented by the addition of E2 (10 ng/ml) relative to that in control cultures, whereas the addition of P4 (100 ng/ml) did not affect EGF-R expression in those cells (Fig. 6). The present study demonstrates the individual effect of ovarian steroids on the proliferative activity of leiomyoma cells cultured in vitro on the basis of PCNA expression. PCNA is a cell cycle-related nonhistone nuclear protein with a molecular mass of 36-kDa. Elevated levels of PCNA appear in late G1 phase and become maximal during the S phase of proliferating cells, but are not detectable in resting cells [13]. Immunohistochemical PCNA labeling has been proven useful in evaluating the proportions of proliferating cells in normal and neoplastic cell populations [14]. In addition to an essential role of PCNA in DNA replication, recent studies suggested an involvement of PCNA in DNA excision repair [25]. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes is affected by PCNA. The data in the present study demonstrate that in leiomyoma cells both E2 and P4 up-regulate the cell-proliferating activity, whereas in normal myometrial smooth muscle cells, only E2 up-regulates the cell proliferating activity [26]. As Eiletz et al. [27] reported, P4 levels in human normal myometrium and leiomyoma tissues were as high as 10 to 70 ng/g protein, whereas E2 levels in human normal myometrium and
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Fig. 6. Effects of E2 and P4 on EGF-R expression in cultured leiomyoma cells as assessed by immunohistochemical analysis. Compared to untreated leiomyoma cells (A), immunostaining for EGF-R was remarkably augmented by treatment with E2 (B), but not by the treatment with P4 (C). Bars ⫽ 5 m. Original magnification, ⫻400.
leiomyoma tissues ranged from 4 to 10 ng/g protein; the concentrations of sex steroids (E2, 10 ng/ml; P4, 100 ng/ml) that were found to be effective in up-regulating cell proliferation in the present study appear to be within the physiological tissue concentration range. Cultured leiomyoma cells had an increased response to P4 compared to that of cultured normal myometrial cells, which is consistent with the reports of Brandon et al. [28] showing that P4 receptor mRNA is overexpressed in uterine leiomyoma compared to that in the adjacent normal myometrium. In the present study, we have also provided the evidence that P4 is capable of increasing the expression of immunoreactive EGF proteins with a higher molecular mass relative to authentic EGF in leiomyoma cells; however, E2 does not have this capability. Up-regulation by P4 of the proliferating activity of leiomyoma cells and the expression of immunoreactive EGF proteins in leiomyoma cells are of great interest, because EGF may be involved in the autocrine/ paracrine regulation of leiomyoma growth. Nelson et al. [16] demonstrated in murine uterine tissues that the effect of E2 may be mediated by EGF and that EGF is capable of replacing E2 in the stimulation of female genital-tract growth. EGF is a 6-kDa polypeptide that is known to be generated by proteolytic processing of a larger molecular precursor, 133-kDa prepro-EGF [29,30]. EGF is shown to be present in its prepro form in the kidney and other tissues [31]. Taking these findings into account, the immunoreactive EGF proteins, with higher molecular masses of 133kDa and 71-kDa induced by P4 treatment in cultured leiomyoma cells, are postulated to be a prepro-EGF-like protein and an active species generated from the preproEGF protein, respectively. The presence of immunoreactive EGF protein and mRNA encoding EGF in human myometrial cells and leiomyoma cells has previously been reported by Rossi et al. [18] using immunohistochemical techniques and by Yeh et al. [17] using PCR, respectively. A potential role for EGF in the regulation of leiomyoma growth is also suggested on the basis of the observations of Lumsden et al.
[32], who demonstrated that the shrinkage of uterine leiomyoma in conjunction with a reduction in E2 levels in serum with GnRH agonist therapy was associated with a remarkable reduction in uterine EGF-binding sites. In this connection, we have noticed in the present study that E2 is capable of increasing the expression of EGF-R in leiomyoma cells, but P4 is not. It is now likely that P4 up-regulates the production of EGF-like proteins in leiomyoma cells, whereas E2 up-regulates the expression of EGF-R in leiomyoma cells. Thus, P4 and E2 seem to participate in leiomyoma growth through the induction of EGF-like proteins and EGF-R expression in leiomyoma cells, respectively. In support of this observation, we demonstrated that not only E2 but also P4 augmented the PCNA protein expression in cultured leiomyoma cells. The fact that P4 upregulates PCNA protein expression in cultured leiomyoma cells supports the in vivo finding of higher PCNA-labeling index in leiomyoma tissues in the secretory, P4-dominated, phase compared to that in the proliferative phase.
4. Bcl-2 protein expression in uterine myometria and leiomyomas throughout the menstrual cycle Apoptosis was first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing, and chromatin condensation culminating in cell fragmentation [33]. Despite the identification of genes necessary for apoptotic cell death, the essential biochemical events in apoptotic cell death remain largely unknown. Korsmeyer [34] reported that the Bcl-2 proto-oncogene was unique among cellular genes in its ability to block apoptotic cell death in multiple contexts. Overexpression of Bcl-2 in transgenic models leads to accumulation of cells due to evasion of normal cell death mechanisms [35]. Accordingly, Bcl-2 is thought to be a cell survival gene. Immunohistochemical examinations of leiomyomas and normal myometrial tissues from the same individual uterus
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Fig. 8. Effects of sex steroids on the expression of Bcl-2 protein in cultured leiomyoma cells as assessed by Western blot analysis. The addition of P4 (100 ng/ml) remarkably increased the 26-kDa Bcl-2 protein expression in the cultured leiomyoma cells compared to that in control cultures, whereas the addition of E2 (10 ng/ml) resulted in somewhat lower expression of the 26-kDa Bcl-2 expression.
Fig. 7. Western blot analysis of leiomyoma and myometrium tissue extracts with a monoclonal antibody to Bcl-2 protein. 26-kDa Bcl-2 protein was abundantly present in leiomyoma tissue extracts, whereas in myometrial tissue extracts Bcl-2 protein was undetectable.
in the proliferative phase of the menstrual cycle demonstrated that Bcl-2 protein was abundantly present in the cytoplasm of leiomyoma cells, but was scarcely present in normal myometrial smooth muscle cells. Comparison between immunostaining for Bcl-2 protein in leiomyoma tissues obtained in the proliferative phase and in the secretory phase showed that Bcl-2 protein expression in the leiomyoma cells in the secretory phase was much more abundant than that in the proliferative phase of the menstrual cycle. By contrast, there was no apparent difference in the intensity of immunostaining for Bcl-2 protein in normal myometrial smooth muscle cells between the proliferative phase and the secretory phase of the menstrual cycle. Western blot analyses of protein extracts from leiomyoma and myometrial tissues revealed that 26-kDa Bcl-2 protein was abundantly present in leiomyoma tissue extracts, whereas in myometrial tissue extracts, Bcl-2 protein was undetectable (Fig. 7). Our present observation that Bcl-2 protein expression is predominant in leiomyoma cells compared to that in normal myometrial cells suggests the possibility that the greater abundance of Bcl-2 protein in leiomyoma cells may be responsible at least in part for the growth of leiomyoma by preventing the apoptotic cell death [36]. In contrast, the scanty expression of Bcl-2 protein in normal myometrial cells raises the possibility that normal myometrial cells may be more susceptible to apoptotic cell death than leiomyoma cells. The increased expression of Bcl-2 protein in leiomyoma cells is likely to be characteristic of leiomyomas and
permits the growth of leiomyomas in the uterus. The enhanced growth of leiomyomas over normal myometrium in vivo may, therefore, be attributed to the increased expression of Bcl-2 protein in leiomyoma cells relative to that in normal myometrial cells.
5. Progesterone in the regulation of Bcl-2 protein expression in leiomyoma cells Effects of sex steroids on Bcl-2 protein expression in leiomyoma cells cultured in vitro were also investigated. Western immunoblotting of proteins extracted from leiomyoma cells cultured for 72 h under serum-free conditions in the absence or presence of E2 or P4 showed that the cultured leiomyoma cells contained immunoreactive Bcl-2 protein with a molecular mass of approximately 26-kDa. The addition of P4 (100 ng/ml) to the serum-free medium remarkably increased the expression of 26-kDa Bcl-2 protein in the cultured leiomyoma cells compared to that in control cultures (Fig. 8). In contrast, the addition of E2 (10 ng/ml) to the serum-free medium resulted in a somewhat lower expression of 26-kDa Bcl-2 protein in the cultured leiomyoma cells relative to that in control cultures (Fig. 8). Unlike the cultured leiomyoma cells, neither treatment with P4 nor treatment with E2 affected the expression of Bcl-2 protein in cultured normal myometrial cells. The fact that Bcl-2 protein expression in cultured leiomyoma cells was remarkably augmented by P4 is consistent with our immunohistochemical observation of higher expression of Bcl-2 protein in leiomyomas in the secretory, P4-dominated, phase compared to that in the proliferative phase. The molecular basis for P4 action in the regulation of leiomyoma growth is not clear, but probably involves the P4-stimulated induction of Bcl-2 protein in leiomyoma cells.
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also have demonstrated greater abundance of Bcl-2 protein in leiomyomas relative to the normal myometrium of the same individual uterus and have shown that the abundant expression of Bcl-2 protein in leiomyoma may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Bcl-2 protein expression in leiomyoma cells predominated in the secretory, P4-dominated, phase of the menstrual cycle compared to that in the proliferative phase. Consistent with these findings, Bcl-2 protein expression in leiomyoma cells cultured in vitro was up-regulated by P4 (Fig. 9). It seems likely, therefore, that P4 may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. References
Fig. 9. Schematic illustration of sex steroidal regulation of leiomyoma growth and apoptosis.
Since Bcl-2 product has been shown to prolong cell survival by preventing apoptotic cell death, P4 may act as a growthpromoting factor in regulating leiomyoma growth through the enhanced inhibition of apoptosis of leiomyoma cells. By contrast, E2 inhibited the induction of Bcl-2 protein in leiomyoma cells. An inverse relationship between Bcl-2 expression and sex steroid hormones has been demonstrated in the endometrium. Up-regulation of Bcl-2 expression by E2 and down-regulation by P4 have been shown in the normal endometrium [37]. Thus, it must be emphasized that the effects of sex steroid hormones on Bcl-2 protein expression vary among the different cell types even in the uterus.
6. Conclusion The molecular mechanism underlying the sex steroidal regulation of myometria and leiomyoma growth has not been defined. We have shown that P4 up-regulates PCNA protein expression in cultured leiomyoma cells. Consistently, PCNA labeling index in leiomyoma tissues predominated in the secretory, P4-dominated, phase of the menstrual cycle compared to that in the proliferative phase. Furthermore, we demonstrated that EGF-like protein expression in the cultured leiomyoma cells was up-regulated by P4, whereas EGF-R expression in those cells was upregulated by E2. As EGF is known to play a crucial role as a local factor in the autocrine/paracrine regulation of leiomyoma growth, it is conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF-like proteins and EGF-R expression in human uterine leiomyoma. We
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Effects of progesterone on uterine leiomyoma growth and apoptosis Takeshi Maruoa,*, Hiroya Matsuoa, Takashi Samotoa, Yosuke Shimomuraa, Osamu Kurachia, Zhijian Gaoa, Yin Wanga, Irving M. Spitzb, Elof Johanssonb a
Department of Obstetrics and Gynecology, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Center for Biomedical Research, The Population Council, New York, NY 10021, USA
Abstract Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. Recently we have found that the use of levonorgestrel-releasing intrauterine system (IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. These clinical experiences prompted us to characterize the effects of progestin on the proliferation and apoptosis of leiomyoma cells cultured in vitro. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either E2 (10 ng/ml) or P4 (100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells; whereas in cultures of normal myometrial cells, the addition of E2 augmented PCNA expression in the cells, but P4 did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P4 treatment resulted in an increase in EGF expression in the cells. In contrast, E2 treatment augmented EGF-R expression in cultured leiomyoma cells, but P4 did not. These results indicate that P4 up-regulates the expression of PCNA and EGF in leiomyoma cells, whereas E2 up-regulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium, suggesting that the abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Furthermore, Bcl-2 protein expression in leiomyoma cells was up-regulated by P4, but down-regulated by E2. Therefore, it seems likely that P4 may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Levonorgestrel-releasing IUS; Progesterone; 17-Estradiol; Uterine myoma; EGF; Bcl-2 protein
1. Introduction Uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium, occurring in as many as 30% of women over 35 years of age [1]. The majority of patients have multiple leiomyomas, and each leiomyoma is thought to be clonal, arising independently from a single smooth muscle cell [2]. Although the nature of the initial event is unknown, a role for ovarian steroid hormones in the growth of uterine leiomyomas is likely, because these tumors grow during the reproductive years, increase in size during pregnancy, and regress after menopause [3,4]. Fur* Corresponding author. Tel.: ⫹81-78-382-6000; fax: ⫹81-78-3826019. E-mail address: [email protected] (T. Maruo).
thermore, treatment with GnRH analogs, which reduces ovarian steroid hormone concentrations, leads to a reduction in the size of leiomyomas; however, enlargement of leiomyomas recurs after therapy with GnRH analogs is discontinued [5]. These findings suggest that leiomyoma growth is dependent on ovarian steroids. On the other hand, a growing body of evidence suggests that the action of estrogen may be mediated in part by local growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I), produced by the target cells [6 – 8]. The mechanisms of action of ovarian steroids in the regulation of leiomyoma growth, however, are not well defined as yet. Homeostatic control of the net growth of tumors is thought to be the result of the dynamic balance between cell proliferation and cell death; too much growth can come
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Fig. l. Hemoglobin levels in blood before and after insertion of LNg-IUS in menorrhagic women with intramural myomas. Bars represent mean ⫾ SD.
from too little death as well as from too much proliferation [9]. Only several years ago, many researchers trying to understand what causes the growth of tumors focused their attention on the pathways within cells that tell them when to divide. However, recent work is showing that cells also have internal pathways that tell them when to die [10]. It is possible that in tumors the death pathway may be suppressed, extending the lives of the cells. Actually, apoptosis, or programmed cell death, is known to occur in tumors either spontaneously or in response to treatment [11,12]. Recently we have found that the use of levonorgestrelreleasing intrauterine system (IUS) (LNg-IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas, because of a striking reduction in menorrhagia. Although some women with large intramural myomas had spontaneous expulsion of LNg-IUS at various intervals, they wanted reinsertion of the device because of remarkable reduction in menorrhagia. Significant increases in hemoglobin levels in blood were obtained after insertion of the devices (Fig. 1). No significant differences were noted in myoma volume and uterine volume, as assessed by MRI examination between pretreatment and 12 months of use. LNg-IUS was proven to be an effective modality for the long-term management of menorrhagia due to uterine myoma and adenomyosis. These clinical experiences prompted us to characterize the effects of progesterone on the proliferation and apoptosis of leiomyoma cells. Thus, we first determined the proliferative activity of leiomyoma cells compared with that of adjacent normal myometrial cells throughout the menstrual cycle by immunohistochemical analysis with a monoclonal antibody to proliferating cell nuclear antigen (PCNA) [13,14]. Furthermore, to understand the role of progesterone in regulating proliferative activity of leiomyoma cells, we examined whether ovarian steroids could influence PCNA expression in leiomyoma cells cultured under serum-free, phenol redfree conditions on the basis of immunocytochemical and immunoblot analyses. Because EGF plays a crucial role as a local factor in regulating leiomyoma growth [15–18],
possible effects of ovarian steroids on the expression of EGF and EGF-R receptor (EGF-R) in the cultured leiomyoma cells also were investigated. On the other hand, recent research efforts have focused on the function of proto-oncogene and tumor suppressor gene products in directing cell fate. In particular, an explosion of research interest has centered around the role of Bcl-2 in controlling the survival and death of cells. It is now evident that the Bcl-2 proto-oncogene encodes a 26-kDa protein that prevents the normal course of apoptotic cell death in a variety of cells induced by tropic factor deprivation or other stimuli without altering proliferation [19 –21]. It seems, therefore, that Bcl-2 protein may play an important role in the growth of tumors. However, to date, no information is available on the expression of Bcl-2 protein in uterine leiomyomas. Thus, we also determined the expression of Bcl-2 protein in leiomyomas in comparison with that in the normal myometrium by means of immunohistochemical techniques and immunoblot analysis with a monoclonal antibody to human Bcl-2 protein. Furthermore, to understand the role of progesterone in regulating the expression of Bcl-2 protein in uterine leiomyomas, we examined whether progesterone could influence the levels of Bcl-2 protein expression in leiomyoma cells cultured in vitro.
2. Proliferative potential of uterine myometria and leiomyomas throughout the menstrual cycle Immunohistochemical examinations of leiomyoma tissues and the adjacent normal myometrial tissues in the proliferative phase of the menstrual cycle demonstrated that PCNA label was positive only in a few normal myometrial cells (Fig. 2A), whereas it was present in a somewhat greater number of leiomyoma cells (Fig. 2B). Immunohistochemical staining for PCNA in those tissues in the secretory phase showed that only a few normal myometrial cell nuclei were positive for PCNA label (Fig. 2C), whereas
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Fig. 2. Immunohistochemical staining of PCNA in sections of normal myometrium (A) and leiomyoma (B) in the proliferative phase, and normal myometrium (C) and leiomyoma (D) in the secretory phase of the menstrual cycle. Bars ⫽ 5 m. Original magnification, ⫻400.
large numbers of the leiomyoma cell nuclei were positive for PCNA label (Fig. 2). Determination of the mean percentage of PCNA-positive nuclei in leiomyoma and the adjacent normal myometrial tissue sections revealed that the PCNA-positive rate was higher in leiomyoma cells than in normal myometrial cells throughout the menstrual cycle (P ⬍ 0.01) and that the PCNA-positive rate of leiomyoma cells was higher in the secretory, progesterone (P4)-dominated, phase than in the proliferative phase (P ⬍ 0.01). There were, however, no differences in PCNA-positive rate in normal myometrial cells in the proliferative phase and in the secretory phase (Fig. 3). The higher PCNA-labeling index in leiomyoma tissues, relative to the adjacent normal myometrial tissues throughout the menstrual cycle, may permit the enhanced growth of leiomyomas over the adjacent normal myometrium in the same uterus. With respect to the participation of P4 in the proliferation of leiomyoma cells, Kawaguchi et al. [22,23] reported that mitotic count in uterine leiomyomas is higher in the secretory phase of the menstrual cycle than in the proliferative phase and suggested that the growth of leiomyoma cells might be dependent not only on the presence of estrogen, but also on P4.
growth has been established [24], estrogen has received much attention as the major factor responsible for leiomyoma development. The mechanism underlying the stimulatory effects of progesterone on leiomyoma growth, however, has not been defined. To investigate the mitogenic effects of progesterone on uterine myometria and leiomyomas, an in vitro culture system of normal myometria and leiomyoma cells was established. Collagenase treatment of normal myometria and leiomyoma tissues provided a pure population of isolated cells with smooth muscle cell char-
3. Progesterone in the regulation of the proliferative potential of uterine myometria and leiomyoma cells Because leiomyoma growth is closely associated with reproductive years, and the vital role of estrogen in uterine
Fig. 3. The mean percentage of PCNA-positive nuclei in leiomyomas and the adjacent normal myometria as assessed by immunohistochemical analysis. Pro, Proliferative phase; Sec, secretory phase. *, P ⬍ 0.01.
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Fig. 4. Effects of E2 and P4 on PCNA protein expression in cultured normal myometrial cells and leiomyoma cells as assessed by Western immunoblot analysis. The 36-kDa PCNA protein was overexpressed in leiomyoma cells compared to normal myometrial cells. In normal myometrial cells, E2 increased the 36-kDa PCNA protein expression, but P4 did not. In leiomyoma cells, not only E2 but also P4 increased the PCNA protein expression in the cells.
acteristics without either stromal or glandular epithelial cell contamination. Immunohistochemical examination of the cultured cells revealed that these cells were immunostained with a monoclonal antibody directed toward the musclespecific protein desmin, but not immunostained with antibodies to either cytokeratin 19, a cytoskeletal protein specific for epithelial cells, or vimentin, a class of intermediate filament protein present in fibroblasts. Western immunoblot analysis showed that both cultured normal myometrial cells and leiomyoma cells contained immunoreactive PCNA with a molecular mass of approximately 36-kDa, and the 36-kDa PCNA expression in leiomyoma cells was more abundant than that in normal myometrial cells in untreated control cultures (Fig. 4). In cultures of normal myometrial cells, the addition of 17estradiol (E2, 10 ng/ml) remarkably increased the 36-kDa PCNA expression in the cells compared to that in control cultures, whereas such a remarkable increase in PCNA expression was not noted with the addition of P4 (100 ng/ml). By contrast, in cultures of leiomyoma cells, treatment with either E2 (10 ng/ml) or P4 (100 ng/ml) increased the 36-kDa PCNA expression in the cells relative to that in control cultures (Fig. 4). Furthermore, Western immunoblot analysis with a monoclonal antibody to EGF revealed that the cultured leiomyoma cells contained immunoreactive EGF with a molecular mass of approximately 133-kDa; the addition of P4 (100 ng/ml) to the serum-free medium resulted in an obvious increase in the level of expression of 133-kDa immunoreactive EGF, together with the appearance of immunoreactive EGF with a molecular mass of approximately 71-kDa, in the cells compared to that in control cultures (Fig. 5). By contrast, the addition of E2 (10 ng/ml) resulted
Fig. 5. Effects of E2 and P4 on EGF-like protein expression in cultured leiomyoma cells as assessed by Western immunoblot analysis. The addition of P4 (100 ng/ml) resulted in a remarkable increase in 133-kDa immunoreactive EGF expression, together with the appearance of 71-kDa immunoreactive EGF; whereas the addition of E2 (10 ng/ml) resulted in a somewhat lower expression of 133-kDa immunoreactive EGF relative to control cultures.
in a somewhat lower expression of 133-kDa immunoreactive EGF in the cells relative to that in control cultures. Instead, EGF-R expression in the cultured leiomyoma cells assessed by immunocytochemical analysis with a monoclonal antibody to EGF-R was notably augmented by the addition of E2 (10 ng/ml) relative to that in control cultures, whereas the addition of P4 (100 ng/ml) did not affect EGF-R expression in those cells (Fig. 6). The present study demonstrates the individual effect of ovarian steroids on the proliferative activity of leiomyoma cells cultured in vitro on the basis of PCNA expression. PCNA is a cell cycle-related nonhistone nuclear protein with a molecular mass of 36-kDa. Elevated levels of PCNA appear in late G1 phase and become maximal during the S phase of proliferating cells, but are not detectable in resting cells [13]. Immunohistochemical PCNA labeling has been proven useful in evaluating the proportions of proliferating cells in normal and neoplastic cell populations [14]. In addition to an essential role of PCNA in DNA replication, recent studies suggested an involvement of PCNA in DNA excision repair [25]. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes is affected by PCNA. The data in the present study demonstrate that in leiomyoma cells both E2 and P4 up-regulate the cell-proliferating activity, whereas in normal myometrial smooth muscle cells, only E2 up-regulates the cell proliferating activity [26]. As Eiletz et al. [27] reported, P4 levels in human normal myometrium and leiomyoma tissues were as high as 10 to 70 ng/g protein, whereas E2 levels in human normal myometrium and
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Fig. 6. Effects of E2 and P4 on EGF-R expression in cultured leiomyoma cells as assessed by immunohistochemical analysis. Compared to untreated leiomyoma cells (A), immunostaining for EGF-R was remarkably augmented by treatment with E2 (B), but not by the treatment with P4 (C). Bars ⫽ 5 m. Original magnification, ⫻400.
leiomyoma tissues ranged from 4 to 10 ng/g protein; the concentrations of sex steroids (E2, 10 ng/ml; P4, 100 ng/ml) that were found to be effective in up-regulating cell proliferation in the present study appear to be within the physiological tissue concentration range. Cultured leiomyoma cells had an increased response to P4 compared to that of cultured normal myometrial cells, which is consistent with the reports of Brandon et al. [28] showing that P4 receptor mRNA is overexpressed in uterine leiomyoma compared to that in the adjacent normal myometrium. In the present study, we have also provided the evidence that P4 is capable of increasing the expression of immunoreactive EGF proteins with a higher molecular mass relative to authentic EGF in leiomyoma cells; however, E2 does not have this capability. Up-regulation by P4 of the proliferating activity of leiomyoma cells and the expression of immunoreactive EGF proteins in leiomyoma cells are of great interest, because EGF may be involved in the autocrine/ paracrine regulation of leiomyoma growth. Nelson et al. [16] demonstrated in murine uterine tissues that the effect of E2 may be mediated by EGF and that EGF is capable of replacing E2 in the stimulation of female genital-tract growth. EGF is a 6-kDa polypeptide that is known to be generated by proteolytic processing of a larger molecular precursor, 133-kDa prepro-EGF [29,30]. EGF is shown to be present in its prepro form in the kidney and other tissues [31]. Taking these findings into account, the immunoreactive EGF proteins, with higher molecular masses of 133kDa and 71-kDa induced by P4 treatment in cultured leiomyoma cells, are postulated to be a prepro-EGF-like protein and an active species generated from the preproEGF protein, respectively. The presence of immunoreactive EGF protein and mRNA encoding EGF in human myometrial cells and leiomyoma cells has previously been reported by Rossi et al. [18] using immunohistochemical techniques and by Yeh et al. [17] using PCR, respectively. A potential role for EGF in the regulation of leiomyoma growth is also suggested on the basis of the observations of Lumsden et al.
[32], who demonstrated that the shrinkage of uterine leiomyoma in conjunction with a reduction in E2 levels in serum with GnRH agonist therapy was associated with a remarkable reduction in uterine EGF-binding sites. In this connection, we have noticed in the present study that E2 is capable of increasing the expression of EGF-R in leiomyoma cells, but P4 is not. It is now likely that P4 up-regulates the production of EGF-like proteins in leiomyoma cells, whereas E2 up-regulates the expression of EGF-R in leiomyoma cells. Thus, P4 and E2 seem to participate in leiomyoma growth through the induction of EGF-like proteins and EGF-R expression in leiomyoma cells, respectively. In support of this observation, we demonstrated that not only E2 but also P4 augmented the PCNA protein expression in cultured leiomyoma cells. The fact that P4 upregulates PCNA protein expression in cultured leiomyoma cells supports the in vivo finding of higher PCNA-labeling index in leiomyoma tissues in the secretory, P4-dominated, phase compared to that in the proliferative phase.
4. Bcl-2 protein expression in uterine myometria and leiomyomas throughout the menstrual cycle Apoptosis was first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing, and chromatin condensation culminating in cell fragmentation [33]. Despite the identification of genes necessary for apoptotic cell death, the essential biochemical events in apoptotic cell death remain largely unknown. Korsmeyer [34] reported that the Bcl-2 proto-oncogene was unique among cellular genes in its ability to block apoptotic cell death in multiple contexts. Overexpression of Bcl-2 in transgenic models leads to accumulation of cells due to evasion of normal cell death mechanisms [35]. Accordingly, Bcl-2 is thought to be a cell survival gene. Immunohistochemical examinations of leiomyomas and normal myometrial tissues from the same individual uterus
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Fig. 8. Effects of sex steroids on the expression of Bcl-2 protein in cultured leiomyoma cells as assessed by Western blot analysis. The addition of P4 (100 ng/ml) remarkably increased the 26-kDa Bcl-2 protein expression in the cultured leiomyoma cells compared to that in control cultures, whereas the addition of E2 (10 ng/ml) resulted in somewhat lower expression of the 26-kDa Bcl-2 expression.
Fig. 7. Western blot analysis of leiomyoma and myometrium tissue extracts with a monoclonal antibody to Bcl-2 protein. 26-kDa Bcl-2 protein was abundantly present in leiomyoma tissue extracts, whereas in myometrial tissue extracts Bcl-2 protein was undetectable.
in the proliferative phase of the menstrual cycle demonstrated that Bcl-2 protein was abundantly present in the cytoplasm of leiomyoma cells, but was scarcely present in normal myometrial smooth muscle cells. Comparison between immunostaining for Bcl-2 protein in leiomyoma tissues obtained in the proliferative phase and in the secretory phase showed that Bcl-2 protein expression in the leiomyoma cells in the secretory phase was much more abundant than that in the proliferative phase of the menstrual cycle. By contrast, there was no apparent difference in the intensity of immunostaining for Bcl-2 protein in normal myometrial smooth muscle cells between the proliferative phase and the secretory phase of the menstrual cycle. Western blot analyses of protein extracts from leiomyoma and myometrial tissues revealed that 26-kDa Bcl-2 protein was abundantly present in leiomyoma tissue extracts, whereas in myometrial tissue extracts, Bcl-2 protein was undetectable (Fig. 7). Our present observation that Bcl-2 protein expression is predominant in leiomyoma cells compared to that in normal myometrial cells suggests the possibility that the greater abundance of Bcl-2 protein in leiomyoma cells may be responsible at least in part for the growth of leiomyoma by preventing the apoptotic cell death [36]. In contrast, the scanty expression of Bcl-2 protein in normal myometrial cells raises the possibility that normal myometrial cells may be more susceptible to apoptotic cell death than leiomyoma cells. The increased expression of Bcl-2 protein in leiomyoma cells is likely to be characteristic of leiomyomas and
permits the growth of leiomyomas in the uterus. The enhanced growth of leiomyomas over normal myometrium in vivo may, therefore, be attributed to the increased expression of Bcl-2 protein in leiomyoma cells relative to that in normal myometrial cells.
5. Progesterone in the regulation of Bcl-2 protein expression in leiomyoma cells Effects of sex steroids on Bcl-2 protein expression in leiomyoma cells cultured in vitro were also investigated. Western immunoblotting of proteins extracted from leiomyoma cells cultured for 72 h under serum-free conditions in the absence or presence of E2 or P4 showed that the cultured leiomyoma cells contained immunoreactive Bcl-2 protein with a molecular mass of approximately 26-kDa. The addition of P4 (100 ng/ml) to the serum-free medium remarkably increased the expression of 26-kDa Bcl-2 protein in the cultured leiomyoma cells compared to that in control cultures (Fig. 8). In contrast, the addition of E2 (10 ng/ml) to the serum-free medium resulted in a somewhat lower expression of 26-kDa Bcl-2 protein in the cultured leiomyoma cells relative to that in control cultures (Fig. 8). Unlike the cultured leiomyoma cells, neither treatment with P4 nor treatment with E2 affected the expression of Bcl-2 protein in cultured normal myometrial cells. The fact that Bcl-2 protein expression in cultured leiomyoma cells was remarkably augmented by P4 is consistent with our immunohistochemical observation of higher expression of Bcl-2 protein in leiomyomas in the secretory, P4-dominated, phase compared to that in the proliferative phase. The molecular basis for P4 action in the regulation of leiomyoma growth is not clear, but probably involves the P4-stimulated induction of Bcl-2 protein in leiomyoma cells.
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also have demonstrated greater abundance of Bcl-2 protein in leiomyomas relative to the normal myometrium of the same individual uterus and have shown that the abundant expression of Bcl-2 protein in leiomyoma may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Bcl-2 protein expression in leiomyoma cells predominated in the secretory, P4-dominated, phase of the menstrual cycle compared to that in the proliferative phase. Consistent with these findings, Bcl-2 protein expression in leiomyoma cells cultured in vitro was up-regulated by P4 (Fig. 9). It seems likely, therefore, that P4 may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. References
Fig. 9. Schematic illustration of sex steroidal regulation of leiomyoma growth and apoptosis.
Since Bcl-2 product has been shown to prolong cell survival by preventing apoptotic cell death, P4 may act as a growthpromoting factor in regulating leiomyoma growth through the enhanced inhibition of apoptosis of leiomyoma cells. By contrast, E2 inhibited the induction of Bcl-2 protein in leiomyoma cells. An inverse relationship between Bcl-2 expression and sex steroid hormones has been demonstrated in the endometrium. Up-regulation of Bcl-2 expression by E2 and down-regulation by P4 have been shown in the normal endometrium [37]. Thus, it must be emphasized that the effects of sex steroid hormones on Bcl-2 protein expression vary among the different cell types even in the uterus.
6. Conclusion The molecular mechanism underlying the sex steroidal regulation of myometria and leiomyoma growth has not been defined. We have shown that P4 up-regulates PCNA protein expression in cultured leiomyoma cells. Consistently, PCNA labeling index in leiomyoma tissues predominated in the secretory, P4-dominated, phase of the menstrual cycle compared to that in the proliferative phase. Furthermore, we demonstrated that EGF-like protein expression in the cultured leiomyoma cells was up-regulated by P4, whereas EGF-R expression in those cells was upregulated by E2. As EGF is known to play a crucial role as a local factor in the autocrine/paracrine regulation of leiomyoma growth, it is conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF-like proteins and EGF-R expression in human uterine leiomyoma. We
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