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Department of Obstetrics and Gynecology (T.M., K.N.), Osaka Prefectural Hospital, Sumiyoshi, Osaka 588 Japan; and Department of Pathology (A.S., N.T.), Hyogo College of Medicine, Hyogo 663 Japan
Abstract
Cell proliferation and apoptosis in uterine leiomyoma were investigated during therapy with GnRH agonist (GnRHa). Patients with uterine leiomyomas were injected with 3.75 mg GnRHa (depot leuprolide acetate) at intervals of 4 weeks and underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, or 16th week of GnRHa therapy. Tissue sections of leiomyomas from these patients and from control patients (control patients received no GnRHa therapy) were stained with the Ki-67 antibody or by an in situ DNA 3'-end labeling method, and numbers of Ki-67 immunostained cells and DNA 3'-end-labeled cells per cm2 were examined as indices of cell proliferation and apoptosis, respectively. The number of Ki-67 immunostained cells/cm2 in leiomyomas at the 2nd week of the GnRHa therapy was comparable with that of control patients. However, it decreased to a level less than one forth that of control patients at the 4th week, and it remained at similar low levels at the 8th, 12th, and 16th week. The number of DNA 3'-end-labeled cells/cm2 in leiomyomas of control patients and in leiomyomas at the 2nd, 8th, 12th, and 16th weeks of GnRHa therapy were at low levels but, at the 4th week, was at an extremely high level (about 5 times more than that of control patients). The present results indicate that GnRHa therapy suppresses cell proliferation and causes a transient increase in apoptosis in uterine leiomyomas.
UTERINE leiomyomas are benign tumors originated from uterine smooth muscle cells. Uterine leiomyomas have estrogen and progesterone receptors, and their growth and maintenance are sensitive to estrogen and/or progesterone; and depletion of these steroids induces their regression (1).
GnRH agonist (GnRHa) inhibits gonadotropin secretion from the pituitary gland by causing pituitary desensitization by down-regulation of GnRH receptors, and GnRHa decreases estrogen and progesterone secretion from the ovary (2). Therefore, therapy of leiomyomas with GnRHa has been tried and has been shown to reduce leiomyoma volume (1, 3, 4, 5, 6). The reduction of tumor volume could be caused by a decrease in cell proliferation, an increase in cell loss by apoptosis or necrosis, and cellular atrophy. However, routine histological examinations of leiomyomas after GnRHa therapy have failed to demonstrate these changes, or their results about these changes contradict each other (7, 8). Moreover, there are no histological examinations in which time courses of these changes were examined. Therefore, the present study was intended to investigate time course effects of GnRHa therapy with depot leuprolide acetate on cell proliferation and apoptosis in uterine leiomyomas.
Materials and Methods
Patients
All procedures were carried out under a protocol approved by the Institutional Review Board of the Osaka Prefectural Hospital, and informed consent was obtained from all patients (61 patients, showing regular menstrual cycles). Their average age ± SEM was 43.6 ± 1.0 (range: 30–49).
Treatment protocol
Sixty-one patients, who were to undergo hysterectomy or myomectomy because of leiomyomas, were divided into 2 groups; a control group (18 patients) and a GnRHa-treatment group (43 patients). Patients in a GnRHa-treatment group were injected sc with 3.75 mg depot leuprolide acetate (Leuprine Depot, Takeda Pharmaceutical Co., Osaka, Japan) at intervals of 4 weeks. Five, 10, 13, 8, and 7 patients underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, and 16th week of GnRHa treatment. Patients in a control group underwent hysterectomy without any medications. One intramural uterine leiomyoma obtained from each patient was fixed in phosphate (0.01 mol/L) buffered 10% paraformaldehyde, and tissue sections of a part of each leiomyoma were prepared. Blood was drawn from control patients, and patients who underwent hysterectomy or myomectomy at the 2nd and 4th week of GnRHa treatment, on the day of the operation, for assay of serum estradiol-17ß (E2).
Immunohistochemistry of Ki-67 antigen
Deparaffinized tissue sections (5-µm thickness), mounted on silane-coated glass slides, were treated with 3% H2O2 in 50% methanol for 30 min. Then, these sections were heated in 10 mmol/L sodium citrate buffer (pH 6.0) for 5 min in a microwave oven (Matsushita Electric Industries, Osaka, Japan) at 600 Watt, four times, to facilitate antigen retrieval. They were sequentially incubated with normal goat serum (1:50 dilution) for 30 min at room temperature, with the rabbit polyclonal antibody (1:100 dilution) for human Ki-67 antigen (Dako Japan, Kyoto, Japan) at 4 C overnight, with biotinylated goat antirabbit IgG (1:200 dilution) (Vector, Peterborough, England) for 30 min at room temperature. The immunoreaction was visualized by an avidin-biotin peroxidase complex method using a Vectastain Elite ABC kit (Vector) with 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO). The sections were lightly counterstained with hematoxylin.
In situ DNA 3'-end labeling
In situ DNA 3'-end labeling was carried out according to the method of Gavrieli et al. (9). Terminal deoxynucleotidyl transferase (Toyobo, Osaka, Japan) and biotin-16-deoxyuridine 5'-triphosphate (Boehringer Mannheim Yamanouchi, Tokyo, Japan) were used at concentrations of 0.2 e.u./µL and 10 µmol/L, respectively. A Vectastain Elite ABC kit (Vector) and 3,3'-diaminobenzidine tetrahydrochloride (Sigma) were used for the detection of biotinylated deoxyuridine 5'-triphosphate incorporated into DNA.
Number of Ki-67 immunostained cells or apoptotic cells in leiomyomas
A part of a tissue section of a leiomyoma on a slide glass was enclosed in ink, and the number of Ki-67-immunostained leiomyoma cells or in situ DNA 3'-end-labeled cells in an enclosed area was counted. Then, an enclosed area was measured with an IBAS image analysis systems (Carl Zeiss, Oberkochen, Germany), and the number of these cells/cm2 was calculated. An average area was 0.975 cm2 (range: 0.756–1.115 cm2).
Assay of serum E2 level
The serum E2 level was determined using an RIA kit (Diagnostic Products Co., Los Angeles, CA). The intra- and interassay coefficients of variation were 6.1% and 6.8%, respectively.
Statistics
Values are presented as means + SEM. Statistical significance was evaluated by one-way ANOVA (P < 0.01).
Results
Serum concentrations (means ± SEM) of E2 of control patients (receiving no GnRHa therapy) and patients at the 2nd and 4th weeks of GnRHa therapy were 109.5 ± 30 (n = 18), 145.5 ± 35 (n = 5), and 20.4 ± 4 (n = 10) pg/mL, respectively. A serum concentration of E2 did not decrease at the 2nd week, but it decreased markedly and significantly at the 4th week. Histologies of the uterine endometrium from these 18 control patients showed that 1 patient was at the proliferative phase and 17 patients at the secretory phase. Thinning of the endometrium, stromal fibrosis, and atrophy of the glands were found in the endometrium of the uteri at the 8th, 12th, and 16th week, but the appearance and extent of these changes in the endometrium at the 4th week varied among patients.
Cell proliferation of leiomyomas during GnRHa therapy was evaluated by
examining the number of Ki-67-immunostained cells/cm2
tissue section of a leiomyoma. Ki-67-immunostained cells were scattered
in a tissue section (Fig. 1A
), and an
average number of Ki-67-immunostained cells/cm2 in
leiomyomas of control patients was about 400 (Fig. 2). The number of Ki-67-immunostained
cells/cm2 in leiomyomas at the 2nd week of GnRHa therapy
was as much as that in leiomyomas of control patients. However, it
decreased to a level less than one forth of the number in leiomyomas of
control patients at the 4th week. The number of Ki-67-immunostained
cells/cm2 at the 8th, 12th, and 16th week remained at low
levels similar to that at the 4th week.
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), and a
frequency in a leiomyoma was evaluated by examining the number of
apoptotic cells/cm2 tissue section of a leiomyoma (Fig. 3).
Apoptotic cells were found in a cluster, rather than scattered. An
average number of apoptotic cells/cm2 in leiomyomas of
control patients was less than 100, and numbers of apoptotic
cells/cm2 in leiomyomas at the 2nd, 8th, 12th, and 16th
week of GnRHa therapy were at levels as low as that in leiomyomas of
control patients. However, the number of apoptotic
cells/cm2 at the 4th week was at an extremely high level,
and an average number was about five times more than that in leiomyomas
of control patients.
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The Ki-67 antigen is a nuclear protein that presents in the G1, S, G2, and M phases but not in the G0 phase of the cell cycle, and it is used as a sensitive index of cell proliferation (10, 11). The present results showed that numbers of Ki-67-stained cells in leiomyomas markedly decreased from the 4th week of GnRHa therapy, indicating that GnRHa reduces cell proliferation of leiomyomas. On the contrary, studies that examined cell proliferation by mitotic figures failed to detect an inhibitory effect of GnRHa on cell proliferation of leiomyomas (7, 8). The difference seems to be ascribed to the difference in sensitivity of the method for estimation of cell proliferation.
Examination of apoptosis by the in situ DNA 3'-end-labeling method revealed a transient increase in apoptosis of leiomyoma cells at the 4th week of GnRHa therapy, and low levels of apoptosis at all times other than this week. This transient increase in apoptosis seems, in part, to be responsible for the early reduction of leiomyoma volume within 1 month of GnRHa therapy (5, 6). A transient increase in apoptosis occurs in the rodent’s uterus after estrogen depletion, and this is responsible for its atrophy (12). On the other hand, the other mechanisms would be responsible for its late reduction after 1 month. Reduction of arterial blood flow of the uterus and leiomyomas caused by hypoestrogenism induced by GnRHa therapy would be one of these mechanisms, as suggested by several studies (1, 5, 6, 13, 14).
A serum concentration of E2 decreased at the 4th week, when the number of Ki-67-immunostained cells per unit area decreased and the number of apoptotic cells per unit area increased. Therefore, the decrease in cell proliferation and the increase in apoptosis seem to be caused by the decrease in a serum concentration of E2. Chegini et al. (15) reported that uterine leiomyomas and myometrium had GnRH receptors, and that GnRHa directly inhibited DNA synthesis of cultured myometrial cells in the presence or absence of estrogen and/or progesterone, suggesting that GnRHa directly suppresses cell proliferation. However, this suggestion seems to be less possible because GnRHa did not suppress cell proliferation until the 4th week of GnRHa therapy.
Hypoestrogenism induced by GnRHa therapy causes adverse effects, such as osteoporosis, vasomotor symptoms, and vaginal atrophy (1). Because of these adverse effects, the duration of GnRHa therapy is limited, and cessation of GnRHa therapy induces regrowth of leiomyomas (1). Therefore, to prevent these adverse effects, GnRHa/steroid add-back therapy has been devised (1). In this therapy, estrogen and/or progesterone, at minimal doses (which do not affect the growth of leiomyomas) are administered to patients during GnRHa therapy. The present results indicate that GnRHa suppresses cell proliferation and induces apoptosis at the 4th week of GnRHa treatment. Therefore, we propose that estrogen should be administered after the 4th week of GnRHa therapy in GnRHa/steroid add-back therapy.
Footnotes
Address all correspondence and requests for reprints to: Takahiro Mizutani, Department of Obstetrics, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594–1101, Japan.
Received September 11, 1997.
Revised December 2, 1997.
Accepted December 9, 1997.
References
This article has been cited by other articles:
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  C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
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N. Chegini and L. Kornberg Gonadotropin Releasing Hormone Analogue Therapy Alters Signal Transduction Pathways Involving Mitogen-Activated Protein and Focal Adhesion Kinases in Leiomyoma Reproductive Sciences, January 1, 2003; 10(1): 21 - 26. [Abstract] [PDF]
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