Increased Expression of Bcl-2 Protein in Human Uterine Leiomyoma ...

0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 1 Printed in U.S.A.

Increased Expression of Bcl-2 Protein in Human Uterine Leiomyoma and Its Up-Regulation by Progesterone* HIROYA MATSUO, TAKESHI MARUO,

AND

TAKASHI SAMOTO

Department of Obstetrics and Gynecology, Kobe University School of Medicine, Kobe, Japan ABSTRACT Uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium. Although Bcl-2 protein is known to be an apoptosis-inhibiting gene product and to prevent apoptotic cell death in a variety of cells, there are no published data regarding whether human leiomyomas express Bcl-2 protein. In the present study, we examined the expression of Bcl-2 protein in leiomyomas in comparison with that in the normal myometrium using an immunohistochemical method and immunoblot analysis with a monoclonal antibody to human Bcl-2 protein. Furthermore, we investigated whether sex steroid hormones could influence the levels of Bcl-2 protein expression in leiomyoma cells cultured in vitro under serum-free, phenol red-free conditions. Immunohistochemical staining for Bcl-2 protein was prominent in leiomyoma cells, but was scarcely present in normal myometrial smooth muscle cells. The expression of Bcl-2 protein in leiomyoma cells was most abundant in the secretory, progesteronedominated, phase of the menstrual cycle, but was less abundant in the

proliferative phase of the menstrual cycle. Western blot analyses of leiomyoma and myometrium tissue extracts revealed that Bcl-2 protein, with a molecular mass estimated at approximately 26 kDa, was abundantly present in leiomyoma tissue extracts, but was undetectable in normal myometrial tissue extracts. In monolayer cultures of uterine leiomyoma cells under a serum-free condition, the addition of progesterone (100 ng/mL) resulted in a striking increase in Bcl-2 protein expression in the cultured leiomyoma cells relative to that in control cultures, whereas the addition of 17b-estradiol (10 ng/mL) resulted in a reduction in Bcl-2 protein expression in the cells. The concentrations of sex steroids used were within the physiological tissue concentrations found in leiomyomas and myometrium. The present results suggest that the abundant expression of Bcl-2 protein may have a molecular basis characteristic of leiomyomas in the human uterus and that progesterone may play a vital role in the enhanced expression of Bcl-2 protein in human uterine leiomyoma cells. (J Clin Endocrinol Metab 82: 293–299, 1997)

U

to be the result of the dynamic balance between cell proliferation and cell death, and too much growth can come 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). Recent research efforts have focused on the function of protooncogene 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. The bcl-2 protooncogene was discovered in lymphoma tumors composed of B cells (13). It is now evident that the bcl-2 protooncogene encodes a 26-kDa protein, localized to mitochondrial and perinuclear membranes. The product of the bcl-2 gene, when elevated in cells either in vivo or in vitro, prevents the normal course of apoptotic cell death in a variety of cells induced by tropic factor deprivation or other stimuli without altering proliferation (14, 15). In addition to extending the life span of certain cells, Bcl-2 protein can promote cell replication by reducing the requirements for growth factors (16, 17). 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 conducted the present studies first to determine the ex-

TERINE leiomyoma is the most common benign smooth muscle cell tumor of the myometrium, occurring in as many as 30% of women over 35 yr of age (1). Uterine leiomyomas are a frequent cause of menorrhagia, dysmenorrhea, pelvic pain, reduced fertility, and recurrent pregnancy loss. The majority of patients have multiple leiomyomas, and each leiomyoma is thought to be clonal, arising independently from a single initiated 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). Furthermore, treatment with GnRH analogs, which reduces ovarian steroid hormone concentrations, leads to a reduction in the size of leiomyomas; however, reenlargement of leiomyomas occurs after therapy with GnRH analogs is discontinued (5). A growing body of evidence suggests that the action of sex steroids may be mediated in part by local growth factors produced by the target cells (6 – 8). However, the mechanisms of ovarian steroid hormone actions in the regulation of leiomyoma growth are not well defined yet. Homeostatic control of the net growth of tumors is thought Received July 11, 1996. Revision received August 19, 1996. Accepted August 28, 1996. Address all correspondence and requests for reprints to: Takeshi Maruo, M.D., Department of Obstetrics and Gynecology, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan. * This work was supported in part by Grant in Aid for Scientific Research 07457388 from the Japanese Ministry of Education, Science, and Culture and the International Committee of the Population Council (New York, NY).

293

294

JCE & M • 1997 Vol 82 • No 1

MATSUO, MARUO, AND SAMOTO

pression 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 ovarian steroids in regulating the expression of Bcl-2 protein in uterine leiomyomas, we examined whether ovarian steroids could influence the levels of Bcl-2 protein expression in leiomyoma cells cultured in vitro under a serum-free, phenol red-free condition. Materials and Methods Materials Phenol red-free DMEM (18, 19) and antibiotic solution (1 3 105 U/L penicillin and 50 mg/L streptomycin) were purchased from Life Technologies (Grand Island, NY). FBS, 17b-estradiol (E2), and progesterone (P4) were obtained from Sigma Chemical Co. (St. Louis, MO). Collagenase was purchased from Wako Pure Biochemical Industry (Osaka, Japan). Monoclonal antibodies to human cytokeratin 19 and human desmin were purchased from Nichirei Corp. (Tokyo, Japan). Monoclonal antibody to human vimentin was purchased from Sigma Chemical Co. (St. Louis, MO). Monoclonal antibody to human Bcl-2 protein was purchased from Dako Corp. (Carpinteria, CA). The 75-cm2 flasks were purchased from Iwaki Glass Corp. (Chiba, Japan).

Tissue collection Uterine leiomyomas and adjacent normal myometrial tissues were obtained from women with regular menstrual cycles who underwent abdominal hysterectomy for medically indicated reasons at Kobe University Hospital. The use of uterine tissues for culture experiments was approved by the institutional review board. The patients ranged in age from 30 – 43 yr with a mean age of 37 yr, and none had received hormonal therapy for at least three cycles before surgery. Informed consent was obtained from each patient before surgery for the use of uterine tissues for the present studies. Samples were excluded if accurate menstrual cycle dates could not be assigned or if unexpected pathology was found (e.g. adenomatous hyperplasia). Each uterine specimen was examined by a pathologist for histological examination and dating of the endometrium. Endometrial tissues were obtained from the extirpated uteri, and the day of the menstrual cycle was determined by endometrial histological dating according to the method of Noyes et al. (20) and the patient’s last menstrual period. A total of 30 uterine leiomyomas and myometrial tissues were collected, of which 11 were from the proliferative phase and 19 from the secretory phase of the menstrual cycle.

Immunohistochemical staining Uterine tissue specimens obtained were fixed in 4% buffered neutral formaldehyde solution, dehydrated, and embedded in paraffin. Sections, 5– 6 mm thick, were deparaffinized and followed by standard histological techniques. Immunohistochemical staining was performed by the avidin/biotin immunoperoxidase method with the use of a polyvalent immunoperoxidase kit (Omnitags, Lipshow, MI) as previously described by Maruo and Mochizuki (21). A mouse monoclonal antibody to human Bcl-2 protein was used as the primary antibody in this study. To assure the specificity of the immunological reaction, adjacent control sections were subjected to the same immunoperoxidase method, except that the primary antibody was replaced by nonimmune murine IgG (Miles, Erkhardt, IN) at the same dilution as the specific antibody. In the control experiments, the replacement of the specific primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining. The intensity of immunostaining was evaluated by repeated staining of the same specimens and by more than two observers. It was graded as (2) for no immunostaining, (1) for weak but definitely detectable immunostaining, (11) for moderate immunostaining, and (111) for intense immunostaining.

Cell culture Uterine leiomyoma tissues and adjacent normal myometrial tissues obtained in the proliferative phase and secretory phase of the menstrual

cycle were, respectively, dissected from endometrial cell layers, washed in PBS, cut into small pieces, and digested in 0.2% collagenase (wt/vol) at 37 C for 3–5 h (22). The leiomyoma cells and myometrial cells were collected by centrifugation at 460 3 g for 5 min and washed several times with DMEM containing 1% antibiotic solution. The isolated leiomyoma cells and myometrial cells were, respectively, plated in 75-cm2 flasks at approximate density of 5 3 105 cells/flask and subcultured for 120 h at 37 C in a humidified atmosphere of 5% CO2-95% air in DMEM supplemented with 10% FBS (vol/vol). The trypan blue exclusion test was used to determine the cell viability. Characterization of the cultured cells was examined using immunostaining with monoclonal antibodies to a muscle-specific protein desmin, to a class of intermediate filament protein present in fibroblast vimentin, and to a cytoskeletal protein for epithelial cells cytokeratin 19. Thereafter, the cultured cells were stepped down to serum-free conditions by incubating in serum-free DMEM in the presence or absence of P4 or E2. Treatment with P4 or E2 was begun when the cultured cells were at approximately 40 –50% confluence, and monolayer cultures were maintained in serum-free DMEM for an additional 72 h.

Protein extraction and Western immunoblotting At the termination of cultures, cultured cells were incubated at 4 C for 15 min in the presence of a lysis buffer consisting of 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mmol/L TrisHCl, and 2 mmol/L phenylmethylsulfonylfluoride, pH 7.5. Cells were subsequently scraped off the plates, the extracts were centrifuged at 13.000 3 g for 30 min, and the supernatants were collected. On the other hand, leiomyomas and adjacent normal myometrial tissues for protein extraction were collected immediately after hysterectomy. These tissue samples were homogenized at 4 C in the above-noted lysis buffer (5 g tissue in 1–2 mL). Homogenates were subsequently centrifuged at 13,000 3 g for 30 min, and the supernatants were collected. Protein estimation of the supernatants was performed by the Bradford assay (23). Each of 150-mg aliquots of proteins extracted from cultured cells and uterine tissues was run on a 10% SDS-polyacrylamide gel under a reducing condition using 20 –25 milliamperes (mA) for the stacking gel and 30 –35 mA for the separating gel for 3– 4 h. The proteins were electrophoretically transferred from gels to nitrocellulose membranes overnight at 30 mA as previously described (24). Blots were exposed overnight to the monoclonal antibody directed against Bcl-2 protein at a dilution of 1:80 in Tris buffer. The antigen-antibody complexes were detected with the secondary antibody using the ECL chemiluminescence detection system (Amersham, Arlington Heights, IL). Control procedures for Western immunoblotting included the substitution of the primary antibody with nonimmune murine IgG and omission of the primary antibody. These controls prevented the appearance of immunoreactive Bcl-2 protein band of the membrane-bound proteins. These experiments were repeated as followed with similar results, and the reported results are representative. Western blot analysis of leiomyoma and myometrium tissue extracts with a monoclonal antibody to Bcl-2 protein were performed six times using six different uterine specimens. Three experiments were performed with uterine specimens obtained in the proliferative phase of the menstrual cycle, and the other three experiments were conducted with uterine specimens obtained in the secretory phase of the menstrual cycle. On the other hand, experiments to investigate the effects of sex steroids on Bcl-2 protein expression in cultured leiomyoma cells were performed four times. Two experiments were performed using leiomyoma cells obtained in the proliferative phase of the menstrual cycle, and the other two experiments were conducted using leiomyoma cells obtained in the secretory phase of the menstrual cycle.

Results

Immunohistochemical examinations of leiomyomas and normal myometrial tissues from the same individual uterus in the proliferative phase of the menstrual cycle demonstrated that Bcl-2 protein was abundantly present in the cytoplasm of leiomyoma cells (Fig. 1A), but was scarcely present in normal myometrial smooth muscle cells (Fig. 1B).

Bcl-2 IN UTERINE LEIOMYOMA

295

FIG. 1. Immunohistochemical staining of formalin-fixed paraffin-embedded sections of leiomyoma (A) and myometrium (B) in the proliferative phase of the menstrual cycle with a monoclonal antibody to Bcl-2 protein. Pronounced immunostaining for Bcl-2 protein was observed in leiomyoma cells, whereas myometrial smooth muscle cells showed scarce immunostaining. Replacement of the primary antibody with nonimmune IgG showed a lack of positive immunostaining for Bcl-2 protein in leiomyoma cells (C). Bars 5 5 mm. Original magnification, 3400.

Replacement of the primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining in the leiomyoma cells (Fig. 1C). Comparison between immunostaining for Bcl-2 protein in leiomyoma tissues obtained in the proliferative phase (Fig. 2A) and for Bcl-2 protein in tissues from the secretory phase (Fig. 2B) 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 (Fig. 2C) and the secretory phase of the menstrual cycle (Fig. 2D). Table 1 summarizes the immunostaining of Bcl-2 protein in leiomyoma and myometrium in the proliferative phase and the secretory phase of the menstrual cycle. Western blot analyses of protein extracts from leiomyoma and myometrial tissues obtained in the proliferative phase of the menstrual cycle revealed that Bcl-2 protein was abundantly present in leiomyoma tissue extracts, whereas in myometrial tissue extracts, Bcl-2 protein was undetectable (Fig. 3). Leiomyoma tissue extracts showed a single immunoreactive band with a molecular mass estimate of approximately 26 kDa. Unlike the leiomyoma tissue extracts, no immunoreactive band was visualized with myometrial tissue extracts, indicating that Bcl-2 protein expression in myometrium was below the sensitivity of the ECL chemiluminescence detection system used. Similar results were obtained with the use of protein extracts of leiomyoma and myometrial tissues obtained in the secretory phase of the menstrual cycle. Collagenase treatment of leiomyoma tissues provided a pure population of isolated cells with smooth muscle cell

characteristics without either stromal or glandular epithelial cell contamination. Immunohistochemical examination of cells cultured for 120 h after collection from leiomyoma tissues revealed that these cells were immunostained with a monoclonal antibody directed toward the musclespecific protein desmin (Fig. 4A), but not immunostained with antibodies to either cytokeratin 19, a cytoskeletal protein specific for epithelial cells (Fig. 4B), or vimentin, a class of intermediate filament protein present in fibroblasts (data not shown). The patterns of immunostaining were consistent, as cultures were monitored with these antibodies regularly. 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 the leiomyoma cells in control cultures (Fig. 5). 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 the leiomyoma cells in control cultures (Fig. 5). There were no apparent differences in the effects of sex steroids on Bcl-2 protein expression between leiomyoma cells obtained in the proliferative phase and those from the secretory phase of the menstrual cycle. 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 (data not shown).

296


JCE & M • 1997 Vol 82 • No 1

FIG. 2. Comparison of immunohistochemical staining for Bcl-2 protein in formalin-fixed paraffin-embedded sections of leiomyoma tissues in the proliferative phase (A) and secretory phase (B) and in myometrial tissues in the proliferative phase (C) and the secretory phase (D). The leiomyoma cells in the secretory phase of the menstrual cycle showed more predominant immunostaining for Bcl-2 protein than the leiomyoma cells in the proliferative phase. However, there was no difference in the intensity of immunostaining for Bcl-2 protein in myometrial smooth muscle cells between the proliferative phase and the secretory phase of the menstrual cycle. Immunostaining for Bcl-2 protein in normal myometrial cells was only slightly observed regardless of the menstrual cycle. Bars 5 5 mm. Original magnification, 3400. TABLE 1. Immunostaining for Bcl-2 protein in leiomyoma and myometrium in the proliferative phase and the secretory phase of the menstrual cycle Menstrual cycle

Proliferative phase (n 5 11)

Secretory phase (n 5 19)

Leiomyoma Myometrium

11 2;1

111 2;1

The results were obtained by examining a total of 30 uteri, 11 from the proliferative phase and 19 from the secretory phase of the menstrual cycle. n represents the number of uteri examined.

Discussion

This study is believed to be the first to demonstrate increased cellular levels of Bcl-2 protein in leiomyoma cells relative to those in normal myometrial smooth muscle cells. The bcl-2 protooncogene was originally identified from a human chromosomal translocation that predisposed affected individuals to malignant transformation of immune cells (25). Since the initial characterization of Bcl-2, numerous studies have reported on the ability of forced Bcl-2 expression to promote the survival of various cell types (26 –28). The

action of Bcl-2 on cellular survival is further exemplified by the ability of human Bcl-2 protein to block apoptotic cell death (29). Apoptosis was first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing, and chromatin condensation culminating in cell fragmentation (30). Despite the identification of genes necessary for apoptotic cell death, the essential biochemical events in apoptotic cell death remain largely unknown. Korsmeyer (31) reported that the bcl-2 protooncogene 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 (32). Accordingly, bcl-2 is thought to be a cell survival gene. Taking these ideas into account, our present observation that Bcl-2 protein expression is predominant in leiomyoma cells compared to that in normal myometrial smooth muscle 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 apoptotic cell death. In contrast, the scanty expression of Bcl-2 protein in normal myometrial

Bcl-2 IN UTERINE LEIOMYOMA

smooth muscle cells raises the possibility that normal myometrial smooth muscle cells may be more susceptible to apoptotic cell death than leiomyoma cells. It is likely that the increased expression of Bcl-2 protein in leiomyoma cells is characteristic of leiomyomas and permits the maintenance and 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. The growth of uterine leiomyomas in vivo has been shown

FIG. 3. Western blot analyses of leiomyoma and myometrium tissue extracts with a monoclonal antibody to Bcl-2 protein. Each 150-mg aliquot of protein extracted from leiomyoma and myometrial tissues obtained in the proliferative phase of the menstrual cycle was subjected to Western blotting as described in Materials and Methods. The proteins were detected with a monoclonal antibody to Bcl-2 protein and an enhanced chemiluminescence detection system. Bcl-2 protein, with a molecular mass estimate of approximately 26 kDa, was abundantly present in leiomyoma tissue extracts, whereas in myometrial tissue extracts, Bcl-2 protein was undetectable.

FIG. 4. Immunohistochemical examination of cells cultured in vitro for 120 h after collection from leiomyoma tissues. The cultured cells were immunostained with a monoclonal antibody directed toward the muscle-specific protein desmin (A), but not immunostained with a monoclonal antibody to a cytoskeletal protein specific for epithelial cells cytokeratin 19 (B). Bars 5 5 mm. Original magnification, 3400.

297

to be dependent on the presence of ovarian steroid hormones. The increased incidence of leiomyomas after the menarche, the enlargement of leiomyomas during pregnancy, and the regression of leiomyomas after the menopause are clinically evident (3, 4). Actually, tissue concentrations of estrogen receptors and P4 receptors have been shown to be higher in leiomyomas than in normal myometrium (33, 34). Tamaya et al. (35) found that the ratio of estrogen receptor level to P4 receptor level was higher in leiomyomas than in normal myometrium and suggested that the relative increase in estrogen receptors in leiomyoma cells may account for an enhancement of their estrogen sensitivity. On the other hand, Kawaguchi et al. (36) reported that the growth of leiomyoma cells was dependent not only on the presence of estrogen, but also on P4 in an in vitro explant culture system. With respect to the participation of P4 in the growth of leiomyomas, mitotic activity in uterine leiomyoma of patients treated with a progestin-only preparation has been shown to be significantly higher than mitotic activity in those receiving a combined estrogen-progestin preparation or that in control subjects (37). It is also noteworthy that mitotic activity in uterine leiomyomas is significantly higher in the secretory, P4-dominated, phase of the menstrual cycle than in the proliferative phase (38). The increased mitotic activity in leiomyomas under the hormonal milieu of P4 dominance suggests that leiomyoma growth may also be affected by P4. In this respect, it is of great interest that treatment with P4 (100 ng/mL) resulted in a striking increase in the expression of Bcl-2 protein in leiomyoma cells cultured under serumfree, phenol red-free conditions, whereas treatment with E2 (10 ng/mL) resulted in a somewhat lower expression of Bcl-2 protein in the cultured leiomyoma cells relative to that in control cultures. As Eiletz et al. (39) reported that P4 levels in human myometrium and leiomyoma tissues were as high as 10 –70 ng/g protein, whereas E2 levels in human myome-

298

JCE & M • 1997 Vol 82 • No 1


FIG. 5. Effects of sex steroids on the expression of Bcl-2 protein in leiomyoma cells cultured in vitro assessed by Western blot analyses. Leiomyoma cells obtained in the proliferative phase of the menstrual cycle were cultured for 72 h in serum-free DMEM in the absence or presence of P4 or E2. Each 150-mg aliquot of protein extracted from cultured leiomyoma cells was subjected to Western blotting. The proteins were detected with a monoclonal antibody to Bcl-2 protein and an enhanced chemiluminescence detection system. The addition of P4 remarkably increased the expression of 26-kDa Bcl-2 protein in the cultured leiomyoma cells compared to that in the leiomyoma cells in control cultures, whereas the addition of E2 resulted in somewhat lower expression of the 26-kDa Bcl-2 protein relative to that in leiomyoma cells in control cultures.

treatment with P4 nor that with E2 affected the expression of Bcl-2 protein, as assessed by Western immunoblot analysis. Furthermore, there was no apparent difference in the immunohistochemically detected cellular levels of Bcl-2 protein in normal myometrial smooth muscle cells between the proliferative phase and the secretory phase of the menstrual cycle. This suggests that no cyclic changes in Bcl-2 protein expression exist in the normal myometrium throughout the menstrual cycle. In conclusion, we have demonstrated greater abundance of Bcl-2 protein in leiomyomas relative to the normal myometrium of the same individual uterus and that 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 under serum-free, phenol red-free conditions was upregulated by P4, but down-regulated by E2. It seems, therefore, likely that P4 may participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells, whereas E2 may participate in leiomyoma growth through the stimulation of proliferative potential of leiomyoma cells (Maruo, T., unpublished data) rather than by inhibition of the apoptosis of leiomyoma cells. 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. References

trium and leiomyoma tissues ranged from 4 –10 ng/g protein, the concentrations of sex steroids used in the present study appear to be within the physiological tissue concentration range. 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 of the menstrual cycle compared to that in the proliferative phase of the menstrual cycle. 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. Since Bcl-2 product has been shown to prolong cell survival by preventing apoptotic cell death (14, 15), it is likely that P4 may act as a growth-promoting factor in regulating leiomyoma growth through the enhanced inhibition of apoptosis of leiomyoma cells. On the other hand, E2 inhibited the induction of Bcl-2 protein in leiomyoma cells. These results suggest that Bcl-2 protein expression in uterine leiomyoma cells may be regulated by sex steroid hormones. To further understand the effects of sex steroid hormones on uterine leiomyoma growth, it will be needed to examine the effects of combined treatment with E2 and P4 on Bcl-2 protein expression in cultured leiomyoma cells. An inverse relationship between Bcl-2 expression and sex steroid hormones has been described in the endometrium. Up-regulation of Bcl-2 expression by E2 and down-regulation by P4 have been shown in the normal endometrium (40). 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. Indeed, in cultured normal myometrial cells, neither

1. Vollenhoven BJ, Lawrence AS, Healy DL. 1990 Uterine fibroids. Br J Obstet Gynaecol. 97:285–298. 2. Buttram Jr VC, Reiter RC. 1981 Uterine leiomyoma: etiology, symptomatology and management. Fertil Steril. 36:433– 447. 3. Rein MS, Nowak RA. 1992 Biology of uterine myomas and myometrium in vitro. In: Barbieri RL. ed. Seminars in reproductive endocrinology. New York: Thieme; 310 –319. 4. Muran D, Gilleson M, Walters JH. 1980 Myomas of the uterus in pregnancy: ultrasonographic follow-up. Am J Obstet Gynecol. 138:16 –19. 5. West CP, Lumsden MA, Lawson S, Williamson J, Baird DT. 1987 Shrinkage of uterine fibroids during therapy with goserelin (Zoladex): a lutenizing hormone-releasing hormone agonist administered as a monthly subcutaneous depot. Fertil Steril. 48:45–51. 6. Huet-Hudson YM, Chakraborty C, De SK, Suzaki Y, Andrews GK, Dey SK. 1990 Estrogen regulates synthesis of epidermal growth factor in mouse uterine epithelial cells. Mol Endocrinol 4:510 –523. 7. Murphy LJ, Ghahary A. 1990 Uterine insulin-like growth factor-1: regulation of expression and its role in estrogen-induced uterine proliferation. Endocr Rev. 11:443– 453. 8. Nelson KG, Takahashi T, Lee DC, Luetteke NC, Mclachlan JA. 1992 Transforming growth factor-a is a potential mediator of estrogen action in the mouse uterus. Endocrinology 131:1657–1664. 9. Wyllie AH, Kerr JFR, Currie AR. 1980 Cell death: the significance of apoptosis. Int Rev Cytol. 68:251–306. 10. Marx J. 1993 Cell death studies yield cancer clues. Science. 259:760 –762. 11. Distelhorst CW. 1988 Glucocorticosteroids induce DNA fragmentation in human lymphoid leukemia cells. Blood. 72:1305–1309. 12. Sarraf CE, Bowen ID. 1986 Kinetic studies on a murine sarcoma, an analysis of apoptosis. Br J Cancer 54:989 –998. 13. Tsujimoto Y, Croce CM. 1986 Structure, transcripts, and protein products of BCL-2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA. 83:5214 –5218. 14. Korsmeyer SJ. 1992 BCL-2 initiates a new categoly of oncogenes: regulators of cell death. Blood. 80:879 – 886. 15. Reed JC. 1994 Bcl-2 and the regulation of programmed cell death. J Cell Biol. 124:1– 6. 16. Reed JC, Talwar HS, Cuddy M, et al. 1991 Mitochondorial protein p26 BCL-2 reduces growth factor requirements of NIH3t3 fibroblast. Exp Cell Res. 195:277–283. 17. Nunez G, London L, Hockenbery D, Alexander M, Mckearn JP, Korsmeyer SJ. 1990 Deregulated BCL-2 gene expression selectively prolongs survival of growth factor deprived hematopoietic cell lines. J Immunol. 144:3602–3610.

Bcl-2 IN UTERINE LEIOMYOMA 18. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS. 1986 Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen responsive cells in culture. Proc Natl Acad Sci USA. 83:2496 –2500. 19. Welshons WV, Wolf MF, Murphy CS, Jordan VC. 1988 Estrogenic activity of phenol red. Mol Cell Endocrinol. 57:169 –178. 20. Noyes RW, Hertig AT, Rock J. 1950 Dating the endometrial biopsy. Fertil Steril. 1:3–25. 21. Maruo T, Mochizuki M. 1987 Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in human placenta: implication for trophoblast proliferation and differentiation. Am J Obstet Gynecol. 156:721–727. 22. Rossi MJ, Chegini N, Masterson BJ. 1992 Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro. Endocrinology. 130:1716 –1727. 23. Bradford MM. 1976 A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem. 72:248 –253. 24. Sakuragi N, Matsuo H, Coukos G, et al. 1994 Differentiation-dependent expression of the BCL-2 proto-oncogene in the human trophoblast lineage. J Soc Gynecol Invest. 1:164 –72. 25. Cleary ML, Smith SD, Sklar J. 1986 Cloning and structual analysis of cDNA for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t (14; 18) translocation. Cell. 47:19 –28. 26. Henderson S, Rowe M, Gregory C, et al. 1991 Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell. 65:1107–1115. 27. Sentman CL, Shutter JR, Hockenberry D, Kanagawa O, Korsmeyer SJ. 1991 Bcl-2 inhibits multiple forms of apoptosis but not negative selction in thymocytes. Cell. 67:879 – 888. 28. Bissonette RP, Echeverri F, Mahboubi A, Green DR. 1992 Apoptotic cell death induced by c-myc is inhibited by bcl-2. Nature. 359:552–554. 29. Hockenbery D, Nunez G, Milliman C, Schreider RD, Koresmyer SJ. 1990

30. 31. 32.

33.

34.

35.

36.

37. 38.

39.

40.

299

BCL-2 is an inner mitochondorial membrane protein that blocks programmed cell death. Nature. 348:334 –336. Kerr JFR, Wylie AH, Currie AR. 1972 Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 26:239 –257. Korsmyer SJ. 1992 bcl-2 initiates a new categoly of oncogenes: regulators of cell death. Blood. 80:879 – 886. Mcdonnell TJ, Deane N, Platt FM, et al. 1989 BCL-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell. 57:79 – 88. Sadan O, van Iddekinge B, van Gelderen CJ. 1987 Oestrogen and progesterone receptor concentrations in leiomyoma and normal myometrium. Ann Clin Biochem. 24:263–267. Otuka H, Shinohara M, Kashimura M, Yoshida K, Okamura Y. 1989 A comparative study of the estrogen receptor ratio in myometrium and uterine leiomyomas. Int J Gynecol Obstet. 29:189 –194. Tamaya T, Fujimoto J, Okada H. 1985 Comparison of cellular levels of steroid receptors in uterine leiomyoma and myometrium. Acta Obstet Gynecol Scand. 64:307–309. Kawaguchi K, Fujii S, Konishi I, Okamura H, Mori T. 1985 Ultrastructual study of cultured smooth muscle cells from uterine leiomyoma and myometrium under the influence of sex steroids. Gynecol Oncol. 21:32– 41. Tiltman AJ. 1985 The effect of progestins on the mitotic activity of uterine fibromyomas. Int J Gynecol Pathol. 4:89 –96. Kawaguchi K, Fujii S, Konishi I, Naubu Y, Nonogaki H, Mori T. 1989 Mitotic activity in uterine leiomyomas during the menstrual cycle. Am J Obstet Gynecol. 160:637– 641. Eiletz J, Genz T, Pollow K. 1980 Sex steroid levels in serum, myometrium, and fibromyomata in correlation with cytoplasmic receptors and 17-beta-hydroxysteroid dehydrogenase activity in different age groups and phases of the menstrual cycle. Arch Gynecol. 229:13–20. Gowpel A, Sabourin JC, Martin A, et al. 1994 Bcl-2 expression in normal endometrium during the menstrual cycle. Am J Pathol. 144:1195–1202.