Molecular Human Reproduction, Vol. 7, No. 11, 1085-1091,
November 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Comparative analysis of cyclin D1 and oestrogen receptor (
and ß) levels in human leiomyoma and adjacent myometrium
1 Department of Obstetrics and Gynecology, Pécs University Medical School, Édesanyák u.13/15, Pécs, H-7624 and 2 Institute of Physiology, Pécs University Medical School, Szigeti u 12, Pécs, H-7643, Hungary
Abstract
The aim of these experiments was to investigate the expression of cyclin D1 and of oestradiol receptors as well as the level of [3H]oestradiol binding in leiomyoma and adjacent myometrium from human uteri at different menstrual phases and at an early stage of menopause. [3H]oestradiol binding was determined by saturation analysis, while the oestradiol receptor (ER) 
and ß and cyclin D1 levels were determined by Western blot analysis of 16 samples of human leiomyomas and corresponding myometria at different hormonal stages. In leiomyomas during all phases of the menstrual cycle, ER expression, high affinity oestradiol binding and cyclin D1 expression were all elevated in comparison with adjacent myometrium. ERß expression and low affinity oestradiol binding were enhanced in leiomyomas only during the proliferative phase. During menopause, ERß expression and low affinity binding were enhanced in leiomyomas, while the ER expression was not significantly enhanced and cyclin D1 levels were similar to that in myometrium. Only the oestradiol binding exhibited any menstrual cycle-related changes. Our data suggest the involvement of cyclin D1 in the growth of leiomyomas during the menstrual cycle. In menopause, there appears to be a switch from ER to ERß expression in leiomyomas, and the induction of cyclin D1 is decreased. The regression of tumour may ensue from these changes at menopause.
cyclin D1/human leiomyoma/oestrogen receptors/menopause/menstrual cycle
Introduction
Uterine leiomyoma is a major public health problem and it may be the most common type of tumour in women. These tumours derive from myometrial cells, enlarge after 30 years of age and regress with menopause. Factors responsible for the initiation and growth regulation of leiomyomas are poorly understood.
Precise control of cellular proliferation is essential for normal development and for prevention of proliferative diseases such as tumours. It is widely known that cell proliferation is strictly regulated by cell-cycle control mechanisms which depend on the activities of different cyclins and cyclin-dependent kinase complexes. Oestrogens, principally oestradiol, are known to be potent mitogens and to regulate cell proliferation in breast, uterus and other proliferating target organs. In cells of these tissues, oestradiol acts in early G1 phase of the cell cycle, as shown by anti-oestrogens that are able to arrest the oestradiol-dependent cancer cells in the G0/G1 phases (Sabbah et al., 1999
). G1 progression and entry into S phase are controlled by cyclins D, E and A. Early in G1 phase, three different D type cyclins (D1, D2 and D3) act as growth factor sensors and their synthesis and assembly with their catalytic subunits, cdk4 and cdk6, are dependent on mitogenic stimulation. In breast cancer cell lines, oestradiol increases the activity of cdk4 and cdk2, induces the expression of cyclin D1 and also decreases the levels of cdk inhibitors (Foster and Wimalasena, 1996; Prall et al., 1997). Similar mechanisms have been observed in the uterus (Altucci et al., 1997). Studies have also shown that cyclin D1 can directly bind to the oestradiol receptors (ER) and enhance transcription of specific genes (Zwijsen et al., 1997).
The age prevalence of leiomyoma suggests the contribution of ovarian steroids in the initiation and control of growth. In recent years, numerous studies have investigated the changes in expression and binding of ER and progesterone receptor in uterine leiomyoma (Brandon et al., 1993, 1995; Rein et al., 1995; Shimomura et al., 1998). The mechanism of leiomyoma growth, and its shrinkage, however, has not been fully understood. A growing body of evidence suggests that the local growth factors, such as epidermal growth factor (EGF), insulin-like growth factor (IGF), opioid peptides produced by the target cells (Shimomura et al., 1998; Környei et al., 1999; Englund et al., 2000; Vértes et al., 2000), are also involved in the pathomechanism of leiomyoma. No data are available concerning the expression of cell cycle regulators and their possible interaction with ER and ERß in human leiomyoma.
The aims of the present experiments therefore were: (i) to investigate the level of cyclin D1 in comparison with that of ER and of oestradiol binding in leiomyoma and adjacent myometrium from human uteri at different menstrual phases; and (ii) to study these parameters at the beginning of leiomyoma regression in the early stage of menopause.
Materials and methods
Tissues
Normal and pathological (leiomyoma) myometrial specimens were obtained from cyclic (n = 12) and menopausal (n = 4) women (aged 38–50 years) undergoing hysterectomy for benign indications with no history of hormonal treatment for at least 3 months before hospitalization. The last menstrual bleeding of menopausal women was 
3 but <12 months earlier.
The Institutional Human Studies Committee approved the use of the tissues and informed consent was obtained from the patients. One tumour was dissected from each uterus. The number of nodules ranged between one and five per patient, and their sizes were 10–50 mm in average diameter. Nodules studied were 35–40 mm in diameter and situated within the myometrial wall of the uterus. Leiomyoma specimens were obtained from the leiomyoma tissue just beneath the capsule of the tumour. Myometrial samples, for use as paired controls, were obtained from surrounding normal myometrium situated >10 mm away from the fibroid capsule. A pathologist examined parts of the selected nodules and all tissue samples used for this study were confirmed as histologically ordinary leiomyoma without any sign of degenerative changes. The uterine samples were also examined by a pathologist to assess the stage of the menstrual cycle and to exclude adenomyosis or malignant changes. The tissue samples after dissection were immediately frozen in liquid nitrogen and were stored at –80°C until use. FSH concentration in the serum of patients was determined by immunoluminometric assay (Byk-Sagtec Diagnostika, Germany) to diagnose the early stage of menopause. Serum concentrations of FSH on morning of hysterectomy were 31 ± 6.2 and 11 ± 8 IU/l (mean ± SD) in menopausal and menstrual cycle patients respectively.
Chemicals
[2,4,6,7-3H]oestradiol (specific activity 3.4 TBq/mmol; MTA Budapest, Hungary) was used for the binding assay. The monoclonal antibody against ER (clone: ER1D5) was purchased from Immunotech (Marseilles, France). For the detection of ERß and cyclin D1 proteins, rabbit polyclonal IgG (Santa Cruz Biotechnology, CA, USA) antibodies were used. All other chemicals, unless stated otherwise, were purchased from Sigma (St Louis, MO, USA).
Radioligand binding assay
The frozen tissue samples were pulverized and then homogenized in TE buffer (10 mmol/l Tris, 1.5 mmol/l EDTA) pH 7.4. Homogenates were centrifuged at 800 g for 20 min. The resulting pellets were resuspended in TE buffer and centrifuged consecutively three times. DNA concentration was determined by a published method (Burton, 1956). Saturation analysis of [3H]oestradiol binding was assessed by an in-vitro oestradiol exchange assay (Környei et al., 1986, 1993; Oszter et al., 2000). Samples (0.2 ml) in triplicates from the resuspended pellets were incubated at 30°C for 60 min with 0.5–30 nmol/l [3H]oestradiol with or without a 1000-fold excess of diethylstilboestrol. After solubilization of binding sites (0°C, 16 h, 0.5 mol/l NaSCN) free ligands were absorbed by dextran-coated charcoal at 0°C for 15 min. Radioactivity was measured in a Packard Tri-Carb 2100TR liquid scintillation analyser. Binding parameters were estimated by a non-linear curve fitting according to the least squares method aided by a computer program developed in our laboratory.
Western blot analysis
The examined tissue samples were homogenized by a Polytron homogenizer at 4°C in 2% sodium dodecyl sulphate (SDS) and 10 mmol/l Tris containing a mixture of protease inhibitors (2.5 µg/ml aprotinin plus 0.3 mmol/l phenylmethylsulphonyl fluoride) at a buffer/tissue ratio of 1 ml/100 mg tissue. Samples were boiled for 5 min and cleared by centrifugation at 10 000 g for 10 min (Lessl et al., 1997). Aliquots of samples were taken for protein determination (Bio-Rad protein assay). The remaining samples were combined with an equal volume of SDS sample-storage buffer (4% SDS, 20% glycerol and 10% mercaptoethanol, in 0.125 mol/l Tris–HCl, pH 6.8), boiled again for 5 min and stored at –20°C.
Proteins (100 µg/ samples) from tissue extracts were separated on a 10% SDS–polyacrylamide mini-gel, and transferred to a nitrocellulose membrane by semi-dry electrophoretic blotting using the Trans-Blot SD cell (Bio-Rad Labs, CA, USA) at 0.8 mA/cm2 for 2 h in a buffer containing 25 mmol/l Tris base, pH 8.5, 0.2 mol/l glycine and 20% methanol. Membranes were incubated for 1 h in blocking solution [phosphate-buffered saline (PBS) containing 5% dried milk, 0.1% Tween 20] and then incubated with primary antibody against ER, ERß, or cyclin D1 protein at a 1:1000 dilution in PBS containing 2.5% dried milk. To ascertain specificity of the stained protein band, the first antibody was preincubated with blocking peptide overnight before being exposed to the membrane. Blots were then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The signals were visualized by an ECL system (Amersham, IL, USA). The intensities of the ER and cyclin D1 protein bands were determined by densitometric scanning. The Western blot analyses were conducted three times in three independent tissue preparations from the same nodules with comparable results. To compare the rate of expression of receptor proteins in different blots, we applied arbitrary units. Proteins were extracted from myometrium of one menopausal uterus, which was used as a standard in all blots. Intensities of the examined bands in different blots were compared to the corresponding intensity of the ‘standard’ myometrium (SM, myometrium from uteri of menopausal women, 6 months after the last bleeding). In each series of experiments, the level of expression in the SM was arbitrarily set to 10 and the results from other examined samples on the same blot were expressed relative to these values.
Statistics
The data are presented as mean ± SD from at least three experiments giving similar results. Group differences were analysed by analysis of variance followed by Student–Newman–Keul's multiple range test. Differences between tissues for the individual groups of patients were calculated by paired t-tests. P < 0.05 was considered statistically significant.
Results
ER and ERß protein expression
The levels of ER in the examined tissues from women during the menstrual cycle and from women at menopause are shown on Figure 1. For all samples analysed, only one molecular species of ER (~65 kDa) reacted specifically with the applied antibody (Figure 1 insert). The ER proteins were elevated, at least by 2-fold, in the extracts from leiomyoma compared to those from myometrium. This increase was not detected in three samples, one in the proliferative phase and two from menopausal uteri (Figure 1). No variation was found in the ER concentration in the examined tissues between the phases of the menstrual cycle and menopause (Figure 1).
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Figure 2
shows the expression of ERß proteins in the same groups of uteri. Expression of ERß was generally weaker than that of ER (Figure 2 insert). By comparing the observed changes to those of the ER, the most striking difference was the very high level of ERß in the menopausal leiomyomas. During the menstrual cycle the level of ERß was also enhanced in leiomyomas during the proliferative phase, but this increase was far less than in the menopausal stage, 32 and 66% increase respectively, relative to the appropriate myometria (Figure 2). The expression of ERß in the myometrium was slightly increased in the secretory phase compared to other phases, but this difference was not statistically significant.
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[3H]oestradiol binding
The [3H]oestradiol binding characteristics of the receptors were determined in the crude nuclear fractions of leiomyoma and adjacent myometria (Környei et al., 1986
). Saturation analysis of [3H]oestradiol binding, in agreement with our former results (Környei et al., 1986), gave two types of binding sites. The type I sites exhibited high affinity (Kd 1.44 ± 0.17 mmol/l) and lower capacity. Scatchard transformation of the data resulted in a linear plot, with the Hill coefficient 
1, indicating competitive binding. The type II binding sites had lower affinity (Kd 21 ± 4.1 mmol/l) and higher capacity. Scatchard transformation of the data was curvilinear, with the Hill coefficient >1, indicating positive cooperativity in the binding (Figure 3).
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The number of high affinity oestradiol sites during the phases of the menstrual cycle was higher in leiomyoma tissues than in samples from adjacent myometrium. During the menopause, however, no difference in the [3H]oestradiol binding was observed between the two types of tissue (Figure 4
). The level of binding to the high affinity sites (type I) in the myometrium was highest during proliferative phase, while these levels were found to be reduced in the other phases to 72–39% of that in the proliferative samples. In the leiomyoma samples, the levels of type I binding were elevated especially in proliferative and menstruation phases, while the lowest binding capacity was detected during menopause (Figure 4).
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The [3H]oestradiol binding to lower affinity sites (Figure 5
) was highest in both tissue types during the proliferative phase, at which time the rate of increase in the leiomyomas was 339 ± 11% compared with the values obtained from myometrium. No further variation in the levels of binding was observed for different stages in the myometrium. However, in the leiomyoma samples from menopausal uteri, at least a 2-fold increase was detected in the type II binding compared with that in the myometrium. It was interesting to note that a similar pattern of changes was observed in ERß expression (Figure 2
).
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Cyclin D1 expression
The expression patterns of cyclin D1 proteins in the samples of the same set of uteri are shown in Figure 6
. The cyclin D1 protein in our samples migrated as a single band with a mol. wt in the 30–35 kDa range. Expression of this protein was detected in all examined tissues, but the rate of expression exhibited marked differences between myometrial and leiomyoma tissues. Cyclin D1 protein was barely detected in the myometria and no fluctuations were observed during the menstrual cycle phases or at menopause. In contrast, in the leiomyoma, the cyclin D1 expression was highly elevated in comparison with that in myometrium during all phases of the menstrual cycle. The rate of increase appeared to be cycle dependent, with the highest difference observed during the proliferative phase and the lowest during the secretory phase (2.5- and 1.7-fold increase relative to the adjacent myometrium respectively). No difference was found between the two types of tissues at menopause.
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Discussion
As the present data show, changes in the levels of both types of ER, of oestrogen binding and of the cyclin D1 protein were observed in uterine leiomyomas in comparison with adjacent myometrium.
These changes indicate a possible role for ER in the pathomechanism of leiomyoma. Uterine leiomyoma derive from myometrial cells, and their growth and development are strongly correlated with the circulatory levels of oestrogen throughout life. Many studies have been devoted to the clarification of the function of ER in the biology of leiomyomas.
The ER are ligand-activated transcription factors that regulate the expression of specific genes by binding to their specific oestrogen-responsive elements (ERE). Until recently it has been generally accepted that only one type of receptor is responsible for the oestrogenic effect in different target cells. However, a new gene encoding a second type of ER, termed ERß, has been described (Kuiper et al., 1996). The ERß protein is smaller than the ER protein, but possesses the modular structure of distinct functional domains. Comparing their protein sequences, considerable homology in the DNA binding domains can be demonstrated, suggesting similar binding characteristics of the receptor to ERE in target genes (Kuiper et al., 1996, 1997). However, the precise functions and regulation of ERß are not known.
The presence of ER in human uterine leiomyoma tissue is well established; however, the literature reveals conflicting data on both ER mRNA and ER proteins in leiomyoma relative to adjacent human myometria. Two studies found no differences in ER mRNA between leiomyoma and myometrium (Vollenhoven et al., 1994; Lessl et al., 1997). By contrast, it has been demonstrated that both mRNA and protein levels of ER are higher in leiomyoma than in the corresponding myometrium (Brandon et al., 1993, 1995; Andersen et al., 1995; Englund et al., 1998). These studies dealt with only the changes of classical ER, at present ER, and much less information is available concerning the changes of the more recently discovered ERß. Expression of ERß mRNA in human myometrium and leiomyoma has been reported (Bennessayag et al., 1999), with elevated ER and ERß mRNA in leiomyoma. However, lower levels of ERß mRNA and higher levels of ER mRNA in leiomyoma relative to that in myometrium in cell culture experiments have been reported (Wu et al., 2000).
In our experiments, both ER and ERß proteins were detected in the examined tissues. The levels of ER were always highest in the leiomyomas. The lower levels of ER proteins in the parent myometrium did not vary with the stages of the menstrual cycle or in menopause. These results seem to contradict another study reporting cycle-dependent changes in ER level in myometrium (Andersen et al., 1995). The differences in data obtained might be caused by anatomical variations in ER concentrations in the myometrium (Richards and Tiltman 1995, 1996). The levels of ERß proteins in our study exhibit only a small variation in the myometrium during the menstrual cycle. In the leiomyoma tissue, the ERß protein levels were elevated during the proliferation stage and most markedly elevated during menopause.
In agreement with previous results from our laboratory (Környei et al., 1986), two types of oestrogen binding sites, with high and low affinities, were observed in human uteri. Since the first demonstration of nuclear oestradiol binding heterogeneity in rat uteri (Eriksson et al., 1978) several data have been reported on type II oestradiol binding sites in uterus (Garai et al., 1985; Vértes et al., 1986). It has been suggested that these sites bind not only oestrogens but also to other, non-oestrogenic ligands and influence, among other processes, cell proliferation in the uterus. Similar to our present results, parallelism has been found between the changes of type II ER binding and the changes in ERß expression in late pregnancy myometria and leiomyoma (Benassayag et al., 1999).
The AP-1 response element seems to be one of the integrators of the transcriptional effects of different hormones (Cato et al., 1992; Bamberger et al., 1996; Uht et al., 1997). The effect of oestrogen on AP-1-driven transcription seems to be ER subtype specific. Oestradiol complexed with ER activates, while when complexed with ERß inhibits, the transcription with AP-1 elements (Paech et al., 1997). On the basis of these results, it could be supposed that the overexpression of ERß at the beginning of the menopause, as shown by our present data, may inhibit the AP-1 DNA binding and result in the changes in transcription of target genes.
Our study also addressed changes in cyclin D1 expression in leiomyomas. Only scattered data are available concerning the role of cyclins in the pathomechanism of leiomyoma growth. Increased sensitivity in expression of cyclin E and cdc2, in parallel with the proliferative effect of HCG in leiomyoma cells, in comparison to that of myometrium has been reported (Horiuchi et al., 2000). In our studies, the levels of cyclin D1 protein were weak in the myometrium, but were greatly enhanced in leiomyoma during the menstrual cycle, while differences were detected between leiomyoma and myometrium during menopause.
As the relevant data show, there is a close relationship between cyclin D1 and ER. Oestradiol increases the expression of cyclin D1 (Prall et al., 1997; Sabbah et al., 1999). Studies on the cyclin D1 gene promoter have identified several regulatory regions including an AP-1 site (Albanese et al., 1995), providing a link between oestrogen-induced AP-1 activity and cyclin D1 expression. The other aspect of this interaction is that the cyclin D1 is able to bind to the hormone binding domain of ER, resulting in its increased binding to ERE so that transcription could be induced in the absence of oestrogen (Zwijsen et al., 1997). No data are available about an interaction between the ERß and cyclins; however, it may be supported by the results found in the present experiments.
To discuss these results is difficult due to lack of data concerning the role of ERß in uterus. It has been reported that the ER ß modulates the transcriptional activity of ER (Hall and McDonnell, 1999). According to these authors, when the circulatory oestradiol level is low, as it is presumed to be during the first year of menopause, the inactive ERß is able to bind to target response elements in a ligand-independent manner and with its repressor domain is able to decrease the overall transcriptional activity of ER. When the oestradiol level increases, the numbers of ligand-activated ER and ERß are elevated, they form heterodimers, block the binding of unliganded ERß, and the oestrogen-specific transcription can precede. Our present results could fit well with this hypothesis.
Our results show that the changes of examined parameters during the menstrual cycle and at menopause are different in the leiomyoma compared to that of adjacent myometrium in human uteri. In leiomyoma, during the menstrual cycle the ER protein, the high affinity oestradiol binding sites and the cyclin D1 expression is elevated. The ERß protein and the low affinity oestradiol binding sites are only higher during the proliferative phase of the menstrual cycle. In menopause, the expression of ERß and the low affinity binding sites are highly enhanced, while the ER protein is only slightly enhanced and the expression of cyclin D1 is not at all enhanced in leiomyoma compared with the myometrium.
In conclusion, this is the first report on elevated in-vivo expression of cyclin D1 in human uterine leiomyoma. Our results suggest that the cell cycle-regulating proteins, at least cyclin D1, are involved in leiomyoma growth during the menstrual cycle, and the reduction of cyclin D1 expression, parallel with the increased concentration of ERß and constant level of ER, may play a role in the regression of leiomyoma in the early period of menopause.
Acknowledgements
This work was supported by the National Science Research Fund (OTKA-T-29267), by the Ministry of Education (FKFP 0494/1999) and by the Ministry of Health (ETT 093/1999) of Hungary.
Notes
3 To whom correspondence should be addressed. E-mail: kovacskalman{at}yahoo.com 
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Submitted on May 10, 2001; accepted on August 16, 2001.
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