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Original Studies |
Department of Obstetrics and Gynecology, Kobe University School of Medicine, Kobe 650, Japan
Address all correspondence and requests for reprints to: Takesi Maruo, M.D., Department of Obstetrics and Gynecology, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
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Abstract |
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Introduction |
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Homeostatic control of the net growth of tumor is the result of the dynamic balance between cell proliferation and cell death (12). It is possible that in tumors the death pathway may be suppressed, extending the lives of the cells (13). We have demonstrated that the abundant expression of Bcl-2 protein, an apoptosis-inhibiting gene product (14, 15), may be one of the molecular bases characteristic of leiomyomas and that progesterone (P4) up-regulates Bcl-2 protein expression in leiomyoma cells (16). Regulation of uterine leiomyoma cell proliferation, however, has been comparatively less studied. Thus, we conducted the present study first to determine the proliferative activity of leiomyoma cells compared with that of adjacent normal myometrial cells throughout the menstrual cycle by immunohistochemical analysis with a monoclonal antibody to proliferating cell nuclear antigen (PCNA) (17, 18). Furthermore, to understand the role of ovarian steroids in regulating proliferative activity of leiomyoma cells, we examined whether ovarian steroids could influence PCNA expression in leiomyoma cells cultured under serum-free, phenol red-free conditions on the basis of immnocytochemical and immunoblot analyses. As EGF has been demonstrated to play a crucial role as a local factor in regulating leiomyoma growth (9, 19, 20, 21, 22), possible effects of ovarian steroids on the expression of EGF and EGF receptor (EGF-R) in cultured leiomyoma cells also were investigated in the present study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phenol red-free DMEM (23, 24) and antibiotics (1 x 105 U/L penicillin and 50 mg/L streptomycin) were purchased from Life Technologies (Grand Island, NY). FBS, 17ß-estradiol (E2), and P4 were obtained from Sigma Chemical Co. (St. Louis, MO). Collagenase was purchased from Wako Pure Biochemical Industries (Osaka, Japan). Monoclonal antibodies to human cytokeratin 19, desmin, and vimentin were purchased from Nichirei Co. (Tokyo, Japan). Monoclonal antibodies to human PCNA and EGF-R were purchased from Calbiochem-Novabiochem International (Uniondale, NY). Monoclonal antibody to human EGF was purchased from Wakunaga Co. (Hiroshima, Japan).
Tissue collection
Uterine leiomyomas and the adjacent normal myometrial tissues were obtained from symptomatic women with regular menstrual cycles who underwent abdominal hysterectomy for medically indicated reasons at Kobe University Hospital. The patients ranged in age from 30–42 yr, with a mean age of 36.4 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 extirpated uterine tissues for culture experiments. 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. (25). A total of 23 uterine leiomyomas, ranging in size up to 7–8 cm, and myometrial tissues were collected from different symptomatic patients, of whom 10 were from the proliferative phase and 13 were from the secretory phase of the menstrual cycle. To control for variations in hormonal milieu between individuals, leiomyoma tissues were compared with the adjacent normal myometrial tissues from the same uterus.
Immunohistochemical staining
Uterine tissue specimens were fixed in 4% buffered neutral formaldehyde solution, dehydrated, and embedded in paraffin. Sections, 5 µm thick, were deparaffinized. Cultured leiomyoma cells were fixed in 90% ethanol. Immunohistochemical staining was performed by the avidin-biotin-immunoperoxidase method with the use of polyvalent immunoperoxidase kit (Omnitags, Lipshow, MI) as previously described (26). Mouse monoclonal antibodies to human PCNA and EGF-R were used at dilutions of 1:80 and 1:100, respectively, as the primary antibody. 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. The replacement of the specific primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining.
Immunostained sections were analyzed in a blinded fashion without knowledge of the experimental group. All stained nuclei were scored as positive for PCNA. The PCNA labeling index was determined by observing more than 1000 nuclei for each experimented samples and was used for evaluating the proliferating activity of the cells. Statistical significance of the difference between the sample means was determined by Student’s t test. A statistically significant difference was considered to be present at P < 0.05.
Cell culture
Uterine leiomyoma tissues and the adjacent normal myometrial tissues obtained from the same individual uterus in the proliferative phase and the secretory phase of the menstrual cycle were, respectively, washed in phosphate-buffered saline, cut into small pieces, and digested in 2% collagenase (wt/vol) at 37 C for 3–6 h (22). The leiomyoma cells and normal myometrial cells were collected by centrifugation at 460 x g for 5 min and washed several times with DMEM containing 1% antibiotic solution. The isolated leiomyoma cells and normal myometrial cells were plated in 75-cm2 flasks at an approximate density of 5 x 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 cell viability. Characterization of the cultured cells was examined using immunostaining with monoclonal antibodies to desmin, vimentin, and cytokeratin 19. As previously described (16), cells cultured for 120 h after collection from leiomyoma tissues were immunostained with the monoclonal antibody to desmin, but were not immunostained with antibodies to either vimentin or cytokeratin 19, indicating a pure population of isolated cells with smooth muscle cell characteristics without either stromal or glandular epithelial cell contamination. Thereafter, the cultured cells were stepped down to serum-free, phenol red-free conditions. Treatment with E2 (0.1, 1.0, and 10 ng/mL) or P4 (1.0, 10, and 100 ng/mL) was begun when the cultured cells were at approximately 30–40% confluence, and monolayer cultures were maintained in serum-free DMEM for an additional 72 h. These cell culture experiments could be performed successfully with six uterine tissue specimens collected from different patients, of which three were from the proliferative phase and the other three were from the secretory phase of the menstrual cycle.
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 Tris-HCl, and 2 mmol/L phenylmethylsulfonylfluoride, pH 7.5. Cells were subsequently scraped off the plates, the extracts were centrifuged at 13,000 x g for 30 min, and the supernatants were collected. Protein estimation of the supernatants was performed by the Bradford assay (27).
Each 100-µg aliquot of proteins extracted from cultured cells was run on a 10% SDS-polyacrylamide gel under reducing conditions. The proteins were electophoretically transferred from gels to nitrocellulose membranes as previously described (28). Blots were exposed either to the monoclonal antibody to PCNA at a dilution of 1:80 or to the monoclonal antibody to EGF at a dilution of 1:100 in Tris buffer. The antigen-antibody complexes were detected with the secondary antibody using the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). Control procedures for Western immunoblotting included substitution of the primary antibody with nonimmune murine IgG and omission of the primary antibody. These controls prevented the appearance of immunoreactive PCNA and EGF bands.
These experiments were performed with six different cultured cell specimens with similar results, and the reported results are representative. Three experiments were performed with the cells obtained in the proliferative phase, and the other three were performed with the cells obtained in the secretory phase of the menstrual cycle.
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Results |
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), whereas it was present in a somewhat
greater number of leiomyoma cells (Fig. 1B). The PCNA label was located
exclusively in the nuclei. Figure 1, D and C, show immunohistochemical
staining for PCNA in leiomyoma tissues and the adjacent normal
myometrial tissues in the secretory phase. Although only a few normal
myometrial smooth muscle cell nuclei were positive for PCNA label (Fig. 1C), a large number of leiomyoma cell nuclei was positive for PCNA
label (Fig. 1D). Replacement of the primary antibody with nonimmune
murine IgG resulted in a lack of positive immunostaining in the
leiomyoma cell nuclei (Fig. 1E).
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).
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shows the PCNA-positive rate of
leiomyoma cells and normal myometrial cells cultured in the absence or
presence of E2 or P4 for
72 h. In cultures of normal myometrial cells, the addition of
E2 (10 ng/mL) increased the PCNA-positive rate of
the cultured cells compared to that in control cultures
(P < 0.001), whereas the addition of
P4 did not. By contrast, in cultures of leiomyoma
cells, the treatment with either E2 (10 ng/mL) or
P4 (100 ng/mL) resulted in significant increases
in the PCNA-positive rate of the cultured cells relative to that in
control cultures (E2 treatment, P
< 0.001; P4 treatment, P <
0.01). The treatment with a lower concentration of either
E2 (0.1, 1.0 ng/mL) or P4
(1.0, 10 ng/mL) did not have a significant effect.
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). In cultures of normal
myometrial cells, the addition of E2 (10 ng/mL)
remarkably increased the 36-kDa PCNA expression in the cells compared
to that in control cultures, whereas such a remarkable increase in PCNA
expression was not noted with the addition of P4.
By contrast, in cultures of leiomyoma cells, treatment with either
E2 (10 ng/mL) or P4 (100
ng/mL) increased the 36-kDa PCNA expression in cells relative to that
in control cultures (Fig. 4).
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). By contrast, the addition of
E2 (10 ng/mL) resulted in a somewhat lower
expression of 133-kDa immunoreactive EGF in the cells relative to that
in control cultures. Instead, EGF-R expression in the cultured
leiomyoma cells assessed by immunocytochemical analysis with a
monoclonal antibody to EGF-R was remarkably augmented by the addition
of E2 (10 ng/mL) relative to that in the cells in
control cultures, whereas the addition of P4 did
not affect EGF-R expression in those cells (Fig. 6). Treatment with lower concentrations
of E2 (0.1 and 1.0 ng/mL) did not augment EGF-R
expression in the cultured cells. There were no apparent differences in
the effects of E2 and P4 on
the levels of immunoreactive PCNA, EGF protein, and EGF-R expression
between leiomyoma cells obtained in the proliferative phase and those
obtained in the secretory phase of the menstrual cycle (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study is believed to be the first to demonstrate that in leiomyoma cells both E2 and P4 up-regulate the cell-proliferating activity, whereas in normal myometrial smooth muscle cells, only E2 up-regulates the cell-proliferating activity without an apparent effect of P4 as assessed by determining the levels of PCNA expression in cultured cells. As Eiletz et al. (34) reported that P4 levels in human normal myometrium and leiomyoma tissues were as high as 10–70 ng/g protein, whereas E2 levels in human normal myometrium and leiomyoma tissues ranged from 4–10 ng/g protein, the concentrations of sex steroids (E2, 10 ng/mL; P4, 100 ng/mL) that were found to be effective in the present study appear to be within the physiological tissue concentration range. The fact that cultured leiomyoma cells had an increased response to P4 compared to cultured normal myometrial cells is consistent with the reports of Brandon et al. (29) showing that P4 receptor messenger ribonucleic acid is overexpressed in uterine leiomyoma compared to that in the adjacent normal myometrium. In the present study, we also provide the first evidence that P4 is capable of increasing the expression of immunoreactive EGF proteins with a higher molecular mass relative to authentic EGF in leiomyoma cells, but E2 is not. Up-regulation by P4 of the proliferating activity of leiomyoma cells and the expression of immunoreactive EGF proteins is of great interest, as it is thought that EGF may be involved in the autocrine/paracrine regulation of leiomyoma growth (19, 20, 21, 22). Nelson et al. (20) demonstrated in murine uterine tissues that the effect of E2 may be mediated by EGF and that EGF is capable of replacing E2 in the stimulation of female genital tract growth. The presence of immunoreactive EGF protein and messenger ribonucleic acid encoding EGF in human myometrial cells and normal myometrial cells has previously been reported by Rossi et al. (22) using immunohistochemical techniques and by Yeh et al. (21) using PCR, respectively. A potential role for EGF in the regulation of leiomyoma growth is also suggested on the basis of the observations of Lumsden et al. (35), who demonstrated that the shrinkage of uterine leiomyoma in conjunction with a reduction in E2 levels in serum with GnRH agonist therapy was associated with a remarkable reduction in uterine EGF-binding sites. In this connection, we have noticed in the present study that E2 is capable of increasing the expression of EGF-R in leiomyoma cells, but P4 is not. EGF is a 6-kDa polypeptide that is known to be generated by proteolytic processing of a larger molecular precursor, 133-kDa prepro-EGF (36, 37). EGF is shown to be present in its prepro form in the kidney and other tissues (38). Taking these findings into account, the immunoreactive EGF proteins with higher molecular masses of 133 kDa and 71 kDa induced by P4 treatment in cultured leiomyoma cells are postulated to be a prepro-EGF-like protein and an active species generated from the prepro-EGF protein, respectively. It is now likely that P4 up-regulates the production of EGF-like proteins in leiomyoma cells, whereas E2 up-regulates the expression of EGF-R in leiomyoma cells. Thus, P4 and E2 seem to respectively participate in leiomyoma growth through the induction of EGF-like proteins and EGF-R expression in leiomyoma cells.
In support of this observation, we demonstrated that not only E2, but also P4, increased the PCNA labeling index of cultured leiomyoma cells and augmented the PCNA protein expression in those cells. The fact that P4 up-regulates PCNA protein expression in cultured leiomyoma cells is in good agreement with the in vivo finding of a higher PCNA labeling index in leiomyoma tissues in the secretory, P4-dominated, phase compared to that in the proliferative phase. Furthermore, we demonstrated that the PCNA labeling index in leiomyoma tissues is significantly higher than that in the adjacent normal myometrial tissues throughout the menstrual cycle. The higher PCNA labeling index in leiomyoma tissues relative to that in the adjacent normal myometrial tissues throughout the menstrual cycle may permit the enhanced growth of leiomyomas over the adjacent normal myometrium in the same uterus. With respect to the participation of P4 in the proliferation of leiomyoma cells, Kawaguchi et al. (39) reported that mitotic count in uterine leiomyomas is higher in the secretory phase of the menstrual cycle than in the proliferative phase and suggested that the mitotic activity of leiomyoma cells may be affected by P4. In this regard, the mitotic activity of uterine leiomyoma in patients treated with a progestin only preparation has been shown to be higher than that in control subjects (40).
In conclusion, we have shown, for the first time, that P4 augments the PCNA labeling index of leiomyoma cells cultured under serum-free, phenol red-free conditions and up-regulates the PCNA protein expression in those cells. Consistent with these in vitro findings, the PCNA labeling index in leiomyoma tissues predominated in the secretory, P4-dominated phase of the menstrual cycle compared to that in the proliferative phase. Furthermore, we also demonstrated that EGF-like protein expression in the cultured leiomyoma cells was up-regulated by P4, whereas EGF-R expression in those cells was up-regulated by E2. As EGF is known to play a crucial role as a local factor in the autocrine/paracrine regulation of leiomyoma growth, it is conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF-like proteins and EGF-R expression in human uterine leiomyoma. Further studies will be necessary to definitively determine the more detailed molecular mechanism by which E2 and P4 interact in the regulation of human uterine leiomyoma growth.
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Footnotes |
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Received September 30, 1997.
Revised February 23, 1998.
Accepted March 3, 1998.
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