Molecular Human Reproduction, Vol. 6, No. 6, 523-528,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
HCG promotes proliferation of uterine leiomyomal cells more strongly than that of myometrial smooth muscle cells in vitro
1 Department of Obstetrics and Gynecology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, and 2 Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Abstract
Uterine myomas often enlarge rapidly during pregnancy. This rapid increase in size may imply that human chorionic gonadotrophin (HCG) influences cell proliferation in uterine leiomyomata. To assess the direct effect of HCG on normal uterine smooth muscle and uterine leiomyomata, we investigated cell proliferation and the expression of cell cycle-related proteins in these cells. Reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed that HCG/LH receptor was present in both cultured myometrial and leiomyomal cells. Treatment with HCG significantly increased cell proliferation in both myometrial and leiomyomal cells (P < 0.03), especially at an early phase in the 9 day culture. The increase in the viable cell number induced by HCG treatment was significantly greater in leiomyoma cells than in myometrial cells on day 3 in culture (P < 0.03). In leiomyomal cells, the expression of proliferating cell nuclear antigen (PCNA), cyclin E and cdc2 was significantly increased by HCG treatment (P < 0.05) even at the lowest concentration used (3 nmol/l). In myometrial cells, the expression of cyclin E and cdc2 was significantly increased by HCG treatment (P < 0.05) only at the highest concentration used (30 nmol/l). These results suggest that HCG directly promotes the proliferation of myometrial and leiomyomal cells, with the latter showing the greater response of the two.
cell proliferation/HCG/HCG/LH receptor/leiomyoma/uterus
Introduction
Uterine leiomyoma is one of the most common human tumours, affecting as many as 30% of women during their reproductive years (Zaloudek and Norris, 1994
). Although the exact aetiology of uterine leiomyoma is as yet unknown, ovarian sex steroid hormones, particularly oestrogen and progesterone, are known to play a major role in the growth of these tumours (Kawaguchi et al., 1989; Gompel and Silverberg, 1992; Brandon et al., 1995). This is supported by clinical studies on uterine myoma (Buttram and Reiter, 1981) and by the presence of oestrogen and progesterone receptors in leiomyomata (Tamaya et al., 1979; Kawaguchi et al., 1991; Englund et al., 1998). However, recent evidence suggests that sex steroid hormones are not the only biologically active substances that stimulate proliferation of uterine leiomyoma cells (Andersen and Barbieri, 1995; Strawn et al., 1995; Van der Ven et al., 1997).
Clinically, uterine myomas often enlarge rapidly during pregnancy (Buttram et al., 1981). Although both oestrogen and progesterone concentrations are elevated during pregnancy, this rapid increase in size may imply that human chorionic gonadotrophin (HCG) also influences cell proliferation in uterine leiomyomata. In fact, the HCG/LH receptor, which is a common transmembrane glycoprotein receptor (Segaloff and Ascoli, 1993), is present in both uterine smooth muscle cells and uterine leiomyomal cells (Singh et al., 1995). In addition, such receptors are also present in the myometrium during pregnancy (Zuo et al., 1994). It has been reported that HCG directly increases both cell numbers and subpopulations of cells by interfering with the cyclic AMP/protein kinase A signalling mechanism in myometrial smooth muscle cells in vitro (Kornyei et al., 1993). Moreover, HCG stimulates prolactin (PRL) secretion in explant cultures of leiomyomata and myometria obtained from premenopausal women and from women treated with leuprolide acetate (Stewart et al., 1994, 1995). In turn, PRL stimulates the proliferation of human leiomyoma cells via the mitogen-activated protein kinase cascade (Nohara et al., 1997). These lines of evidence suggest that HCG may play a definite role in the proliferation of uterine smooth muscle cells and leiomyomal cells. However, the effects of HCG on myometrial or leiomyomal cells have not yet been analysed, and possible differences between the responses to HCG administration shown by normal smooth muscle and leiomyomal cells have not been examined.
In recent years, it has become clear that cell proliferation is regulated by a variety of cell cycle-related substances such as cyclins and cyclin-dependent kinases (cdks). Cell proliferation requires the formation of a complex between a specific cyclin and the corresponding cdk that promotes cell cycling (Nurse, 1994; Sherr and Roberts, 1995). Also important is proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerase 
that is expressed throughout the cell cycle. Although the expression of cyclins, cdks and PCNA has been demonstrated in cells that are actively proliferating, these cell cycle-related proteins has not been evaluated in myometrial and leiomyomal smooth muscle cells. The effect of HCG on cyclins, cdks and PCNA expression remains to be clarified. In the present study, we set out to examine cell proliferation and the expression of cell cycle-related proteins with and without HCG in cultured myometrial and leiomyomal cells.
Materials and methods
Cell culture and HCG treatment
Normal myometrium and leiomyoma were both obtained from seven premenopausal women with uterine leiomyoma who underwent hysterectomy (aged 36–42 years). All of the women menstruated regularly and none had received any hormonal treatment. The tissues were used after obtaining written consent from the patient. The phase of the menstrual cycle was determined by endometrial dating (Noyes et al., 1950): three were in the proliferative phase and four in the secretory phase of the menstrual cycle. Cell culture was performed as described previously (Horiuchi et al., 1999). Briefly, collected tissue was minced into fine pieces in Dulbecco's modified Eagle's medium (DMEM) (Sigma, St Louis, MO, USA) containing 10% fetal bovine serum (FBS; Intergen, NY, USA) and 1% antibiotic–antimycotic solution (Gibco, Grand Island, NY, USA), and the tissues were treated with 0.4% collagenase (Wako, Osaka, Japan) in DMEM at 37°C for 4 h with continuous mixing. The cell suspension was diluted in an equal volume of calcium- and magnesium-free Dulbecco's phosphate-buffered saline (PBS; Gibco), and then centrifuged. The cell pellet was resuspended in DMEM at a concentration of 4x104 cells/ml, and primary culture proceeded at 37°C in 5% CO2 in air for 3–4 days. The primary cultured cells were immunostained for 
-smooth muscle actin (Dako, Glostrup, Denmark) to confirm their smooth muscle origin (Nowak et al., 1993). One part of the population of primary cultured cells was used for reverse transcription–polymerase chain reaction (RT–PCR) analysis for the HCG/LH receptor. The primary cultured cells were transferred to 2.0 cm tissue-culture plates (2x104 cells/plate) for the cell proliferation assay and to a 75 cm2 flask (4x104 cells/ml) for Western blotting analysis. The medium was changed to Phenol Red-free DMEM (Gibco) containing 10% charcoal-striped FBS for the secondary culture to avoid the effect of steroids. Highly purified HCG (3000 IU/mg) (Funakoshi, Tokyo, Japan) was first added to the plates at a concentration of 30 nmol/l (for the cell proliferation analysis) and to the flasks at concentrations of 3, 10 and 30 nmol/l (for Western blotting) at 24 h (the time the medium was changed) in the secondary culture. The concentrations of HCG were selected on the basis of a previous report (Kornyei et al, 1993). HCG was then added every 24 h for 9 days until just before the confluent phase (to avoid contact inhibition).
RT–PCR for HCG/LH receptor
Total RNA was extracted by the acid guanidinium–phenol–chloroform method as described previously (Chomczynski and Sacchi, 1987; Horiuchi et al., 1998). Human ovarian and intestinal tissues were used for the controls. After 1 µg of total RNA had been treated with 1 IU/10 µl DNase I (Life Technologies, Gaithersburg, MD, USA), an RT–PCR assay was performed using an RNA PCR Kit (Takara Shuzo, Ohtsu, Japan), the RNA sample being added to 20 µl of a reaction mixture consisting of 10 mmol/l Tris–HCl (pH 8.3), 50 mmol/l KCl, 5 mmol/l MgCl2, 1 mmol/l dNTP mixture, 1 IU/µl of RNase inhibitor, 0.25 IU/µl of AMV reverse transcriptase, and 0.125 µmol/l of oligo d(T)-adaptor primer. Using a thermal cycler (Perkin Elmer, Gene Amp PCR System 2400-R, Norwalk, CT, USA), the reaction mixture was incubated at 42°C for 30 min, heated at 99°C for 5 min, and then cooled down to 5°C for 5 min. The PCR amplification was carried out by adding 80 µl of PCR reaction mixture containing 10 mmol/l Tris–HCl (pH 8.3), 50 mmol/l KCl, 2.5 units/100 µl of TaKaRa Taq DNA polymerase, and 0.2 µmol/l of a set of 20–22-mer oligonucleotide primers either for HCG/LH receptor cDNA or for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA (to confirm the integrity of the RNA). Primers were synthesized to encompass a specific segment of the cDNA sequence of the HCG/LH receptor (Minegishi et al., 1990) (sense, 5'-TTAATGG CTACCAATAAAGAT-3' and antisense, 5'-CAATCCACCTTGAAGTTGTCCA-3', spanning 373 bp in exons 11) or of G3PDH (sense, 5'-ACGACCACTTTGTCAAGCTC-3' and antisense 5'-TCACAGTTGCCATGTAGACC-3', spanning 226 bp between exons 7 and 8). The corresponding cDNA fragments were denatured at 94°C for 1 min, annealed at 58°C for 1 min, and extended at 72°C for 2 min. After 35 cycles of amplification, the PCR products were analysed on a 2% agarose gel, and the bands were visualized using ethidium bromide during exposure to an UV transilluminator.
Analysis of cell proliferation
Cell proliferation of the cultured myometrial and leiomyomal cells was evaluated by counting the number of viable cells. After 3, 6 and 9 days of secondary culture with or without HCG (at a concentration of 30 nmol/l), the cells were treated with trypsin and resuspended in PBS. The cells were stained with 0.2% Trypan Blue (Nalgene, Dainippon Pharmaceutical, Tokyo, Japan) and the number of viable cells was counted in a haemocytometer chamber. Six or more plates were examined for each of days 3, 6 and 9.
Western blotting
For the Western blotting, cultured cells were retrieved after 9 days in secondary culture (to obtain a sufficient quantity of protein). Cells were lysed in a buffer containing 50 mmol/l Tris–HCl, pH 8.0, 0.25 mol/l NaCl, 0.5% NP-40, 1 mmol/l phenyl methyl sulphonyl fluoride (PMSF; Sigma), 1 mg/ml aprotinin (Boehringer Mannheim, Germany), 1 mg/ml leupeptin (Boehringer Mannheim), 20 mg/ml TPCK (Boehringer Mannheim). The lysates were centrifuged at 13 000 g for 20 min at 4°C and the supernatants were stored at –80°C. Extracts equivalent to 30 µg of total protein were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% acrylamide) and transferred onto nitrocellulose membranes (Hybond TM-C super, Amersham). The membranes were blocked in TBST (0.2 mmol/l NaCl, 10 mmol/l Tris–HCl, pH 7.4, 0.2% Tween-20) containing 5% non-fat dry milk and 0.02% NaN3 for 1 h. This was followed by incubation first with specific antibodies against PCNA (Dako), cyclin E, cdk2, cdc2 (Santa Cruz Bio Inc, Santa Cruz, CA, USA) and ß-actin (Biomakor, Rehovot, Israel) diluted 1:500 in TBST, and second with anti-mouse immunoglobulin (for PCNA, cyclin E, cdc2 and ß-actin) or anti-rabbit immunoglobulin (for cdk2) (Amersham) diluted 1:1000 in TBST. Bound antibody was detected using an enhanced chemiluminescence (ECL) system (Amersham). The density of the bands on the filters was quantified by densitometric analysis using a Quantity One Scan System (ATTO, Tokyo, Japan).
Statistical analyses
Two-factor (treatment with HCG and duration of culture) factorial ANOVA (analysis of variance) was used to compare the number of viable cells during culture in myometrial and leiomyomal cells. Mann–Whitney's U-test was used to compare myometrial and leiomyomal cells in terms of the increase in the viable cell number induced by HCG treatment. The Kruskal–Wallis test and the Bonferroni–Dunn test were used to compare the density of the bands in Western blotting.
Results
Expression of HCG/LH receptor mRNA
In both myometrial and leiomyomal cells, a specific band for HCG/LH receptor mRNA was detected at the 373 bp predicted from the specific primer set used (Figure 1). A specific band for G3PDH mRNA was also detected at the predicted 226 bp in all the specimens (Figure 1).
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Analysis of cell proliferation
In cultures of myometrial and leiomyomal smooth muscle cells, the number of viable cells showed a significant increase during the culture period both in HCG-treated and control cultures (P < 0.001 in each case), though the increases were significantly greater in HCG-treated cultures than in the controls (P = 0.0019 for smooth muscle cells, Figure 2a
; P = 0.0003 for leiomyomal cells, Figure 2b). The effect on the viable cell number induced by HCG treatment at a given time-point was evaluated by expressing the number of viable cells in HCG-treated culture as a percentage of the number in control (non-HCG-treated) culture (Figure 2c). This analysis showed that the effect of HCG was significantly greater for leiomyomal cells than for myometrial cells on day 3 (P = 0.028), while on day 6 (P = 0.91) and day 9 (P = 0.082) the difference was not significant. The number of viable cells generally showed a wider distribution in HCG-treated cultures than in the controls. The size of the increase in the viable cell number showed no correlation with the phase of the menstrual cycle.
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Expression of cell cycle-related proteins
Specific bands for PCNA, cyclin E, cdk2 and cdc2 were detected in both myometrial and leiomyomal cells at 36, 50, 33 and 34 kDa respectively (Figure 3
). ß-actin was expressed at 42 kDa in all the samples. In leiomyomal cells, the band density were significantly increased (compared with controls) as follows: at 10 and 30 nmol/l HCG for PCNA (P = 0.04 and P = 0.018 respectively); at 3, 10 and 30 nmol/l HCG for cyclin E (P = 0.049, P = 0.04, and P = 0.0495 respectively); and at 3, 10 and 30 nmol/l HCG for cdc2 (P = 0.038, P = 0.05, and P = 0.03 respectively) (Figure 4). No significant difference was found in the density of the band for cdk2 between HCG-treated cells and the controls in leiomyomal cells. In myometrial cells, the cell band and densities were significantly increased (compared with controls) only at 30 nmol/l HCG for cyclin E (P = 0.021) and at 30 nmol/l HCG for cdc2 (P = 0.02, Figure 4). No significant difference was found in the density of the bands for PCNA or cdk2 between HCG-treated cells and the controls.
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Discussion
We have confirmed that HCG/LH receptor mRNA is transcribed in both myometrial and leiomyomal cultured cells, a finding consistent with previous reports (Reshef et al., 1990; Singh et al., 1995). Although the HCG/LH receptor was once thought to be present only in gonadal tissues, it has since been found in other human reproductive tissues, e.g. uterus, decidua, placenta (Reshef et al., 1990), fetal membrane (Toth et al., 1996), Fallopian tube (Lei et al., 1993a), umbilical cord (Rao et al., 1993), and uterine artery (Toth et al., 1994). This wide distribution of HCG/LH receptors suggests that HCG may be able to exert a direct effect on a variety of female genital tissues. In addition, HCG/LH receptors have also been demonstrated in pathological conditions, e.g. endometriosis, adenomyosis, and endometrial carcinoma, and in ovarian cancer (Lincoln et al., 1992; Lei et al., 1993b; Lin et al., 1994; Mandai et al., 1997). Thus, HCG may be involved in the regulation of cell proliferation not only in normal tissues, but also in proliferating lesions of the female reproductive tract.
In the present study, we found that HCG induced proliferation of both myometrial cells and leiomyomal cells in vitro, a result consistent with a previous finding (Kornyei et al., 1993). When the viable cell number in HCG-treated culture was expressed as a percentage of that in the non-HCG-treated control at the same time-point, the effect of HCG was seen to be greater on day 3 in culture than on days 6 or 9 for both myometrial and leiomyomal cells. Thus, the effect of HCG on cell proliferation appears to be at its greatest in the early phase of the culture and to then decrease as the culture period progresses. Moreover, the effect on cell proliferation induced by HCG treatment was significantly greater in leiomyomal cells than in myometrial cells in the early phase (day 3) of the culture. The difference between myometrial and leiomyomal cells seems to indicate that the regulatory mechanism governing cell proliferation may not be quite the same in these two cell types. On the other hand, different expression levels of the functional HCG/LH receptor may have been responsible for the different responses to HCG between cultured myometrial and leiomyomal smooth muscle cells. It has been reported that in vivo human uterine leiomyomata express a functional HCG/LH receptor gene at a reduced level compared with that found for the corresponding normal myometrium (Singh et al., 1995). Since we have not quantified the expression level of the HCG/LH receptor between cultured myometrial and leiomyomal smooth muscle cells, further analysis of the expression levels of this receptor will be necessary before we can completely understand the functional importance of the effects of HCG on leiomyoma. Simultaneously, such an analysis might reveal whether a decrease in HCG/LH receptor expression occurs during cell culture; if so, this could explain why HCG increases cell proliferation particularly in the early phases of in-vitro growth.
We also demonstrated that the expression of PCNA, cyclin E and cdc2 was all significantly increased in leiomyomal cells. In particular, the expression of cyclin E and cdc2 were both significantly increased even at the lowest concentration (3 nmol/l) of HCG used. This significant induction of cyclin E and cdc2 by HCG in leiomyomal cells indicates that HCG, even at a low concentration, may exert a direct influence over the growth of uterine myomas by promoting cell cycling. In normal myometrial cells, on the other hand, an increase in protein expression was observed for cyclin E and cdc2 only at 30 nmol/l HCG; no effect at all was seen at lower concentrations (3 and 10 nmol/l). Thus, the mechanism responsive to HCG with respect to cell proliferation may differ between leiomyomal and myometrial cells, although the exact nature of this difference has yet to be explained. It may be that cell proliferation is regulated in a different way between leiomyomal and myometrial cells; the difference seen in the effects of HCG would be a reflection of this phenomenon. However, the details of the biological effects of HCG have not yet been fully characterized, and the effects of HCG on myometrial and leiomyomal cells will certainly require further investigation.
Clinically, gonadotrophin-releasing hormone (GnRH) analogues are used for the treatment of uterine leiomyomata. Their effectiveness is believed to rely on the reduced oestrogen level that results from the induced decrease in gonadotrophins (Friedman et al., 1989; Kettel et al., 1993; Oguchi et al., 1995). We earlier reported that, in leiomyoma cells, the GnRH receptor was present (Kobayashi et al., 1997), as also reported by Wiznitzer et al. (1988), and that a GnRH analogue suppressed cell proliferation (Kobayashi et al., 1997). Thus, the reduced LH concentration during GnRH analogue therapy may provide uterine leiomyoma patients with an additional therapeutic benefit. Be that as it may, GnRH and HCG/LH both seem to have a direct effect on cell proliferation in uterine leiomyomata, and changes in GnRH and HCG/LH concentrations may therefore modulate the therapeutic effect of GnRH analogues in patients with leiomyoma. Moreover, since HCG shows considerable homology with LH, further investigation of the effect of LH on myometrial and leiomyomal cells would seem to be necessary.
Acknowledgments
This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (No. 08457438, 09877318, 09671671), Japan.
Notes
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Shinshu University School of Medicine, 30-1-1 Asahi, Matsumoto 390-8621, Japan. E-mail: tnikaido{at}hsp.md.shinshu-u.ac.jp 
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Submitted on November 15, 1999; accepted on March 10, 2000.
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D. D. Baird, J. S. Kesner, and D. B. Dunson Luteinizing Hormone in Premenopausal Women May Stimulate Uterine Leiomyomata Development Reproductive Sciences, February 1, 2006; 13(2): 130 - 135. [Abstract] [PDF]
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K. A. Kovacs, A. Oszter, P. M. Gocze, J. L. Kornyei, and I. Szabo Comparative analysis of cyclin D1 and oestrogen receptor ({alpha} and {beta}) levels in human leiomyoma and adjacent myometrium Mol. Hum. Reprod., November 1, 2001; 7(11): 1085 - 1091. [Abstract] [Full Text] [PDF]
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O. Kurachi, H. Matsuo, T. Samoto, and T. Maruo Tumor Necrosis Factor-{{alpha}} Expression in Human Uterine Leiomyoma and Its Down-Regulation by Progesterone J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2275 - 2280. [Abstract] [Full Text]
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