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Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. In order to characterize the molecular mechanism of sex steroidal regulation of leiomyoma growth, we examined whether sex steroids could influence the proliferation of leiomyoma cells. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either estradiol (E₂; 10 ng/ml) or progesterone (P₄; 100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells, whereas in cultures of normal myometrial cells, the addition of E₂ augmented PCNA expression in the cells, but P₄ did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P₄ treatment resulted in an increase in EGF expression in the cells, whereas E₂ treatment resulted in a lower EGF expression in the cells. By contrast, E₂ treatment augmented EGF-R expression in cultured leiomyoma cells, but P₄ did not. These results indicate that P₄ upregulates the expression of PCNA and EGF in leiomyoma cells, whereas E₂ upregulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P₄ and E₂ act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium and that Bcl-2 protein expression in leiomyoma cells was upregulated by P₄, but downregulated by E₂. It seems, therefore, likely that P₄ may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. The abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of a leiomyoma relative to that of normal myometrium in the uterus.

Objective: To elucidate the direct effects of gonadotropin-releasing hormone agonist (GnRHa) on the growth of human uterine leiomyoma cells, cell proliferation and apoptosis in cultured leiomyoma cells treated with GnRHa were investigated. Methods: Isolated leiomyoma cells were subcultured in DMEM supplemented with 10% FBS for 5 days and stepped down to serum-free conditions for an additional 6 days in the presence or absence of graded concentrations of GnRHa (10 29 mol/l to 10 212 mol/l). The effects of GnRHa on the number of viable cells, expression of proliferating cell nuclear antigen (PCNA), Fas and Fas ligand, and apoptosis in cultured leiomyoma cells were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) assay, immunocytochemical analysis, Western blot analysis and TUNEL assay respectively. RT-PCR was performed to detect the expression of GnRH receptor mRNA in cultured leiomyoma cells. Results: Treatment with GnRHa resulted in a decrease in the number of cultured viable leiomyoma cells assessed by MTT assay in a dose-dependent manner compared with that in control cultures ðP , 0:01Þ: The growth inhibition of cultured leiomyoma cells treated with GnRHa in concentrations higher than 10 210 mol/l was associated with the suppression of the proliferative potential characterized by a decrease in PCNA-positive rate of the cultured cells ðP , 0:01Þ and an increase in the apoptosis-positive rate assessed by TUNEL assay ðP , 0:05 and P , 0:01Þ: GnRHa markedly increased the expression of Fas and induced the expression of Fas ligand in the cultured leiomyoma cells on the basis of Western blot analysis. These direct effects of GnRHa on the number of viable cultured leiomyoma cells, PCNA-positive rate, apoptosis-positive rate and Fas/Fas ligand expression in the cultured leiomyoma cells were only attained after the 4-day treatment. RT-PCR analysis revealed that GnRH receptor mRNA was expressed in cultured leiomyoma cells. Conclusions:The present results demonstrate that GnRHa directly inhibits the growth of human uterine leiomyoma cells by suppressing cell proliferation and inducing apoptosis, which might be associated with the increase in Fas expression and the induction of Fas ligand expression in the cells.

The present study provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured leiomyoma cells without affecting IGF-I receptor mRNA expression.

Although tumor necrosis factor-alpha (TNFalpha) has been shown mainly to inhibit proliferation and induce apoptosis in a variety of cells, no information is available regarding whether human leiomyoma cells express TNFalpha. In the present study, we examined the expression of TNFalpha in leiomyomas, in comparison with that in the adjacent normal myometrium, using immunohistochemical staining and Western immunoblot analysis with a polyclonal antibody to human TNFalpha. Furthermore, we investigated the effect of sex steroid hormones on TNFalpha expression in leiomyoma cells cultured under serum-free, phenol red-free conditions. Immunohistochemical staining showed that TNFalpha expression in leiomyoma cells was higher than that in the adjacent normal myometrial cells, being more abundant in the proliferative phase than in the secretory, progesterone (P4)-dominated, phase of the menstrual cycle. TNFalpha expression in leiomyoma cells in pregnant uterus was scarce. Western immunoblot analyses of leiomyoma and normal myometrial tissue extracts revealed that TNFalpha, with a molecular mass of 17.3 kDa, was abundantly present in leiomyoma tissue extracts, relative to normal myometrial tissue extracts, and that TNFalpha expression in leiomyoma cells was most abundant in the proliferative phase of the menstrual cycle, less abundant in the secretory phase, and least abundant in pregnant uterus; whereas no such changes in TNFalpha expression were noted in the normal myometrium. In monolayer cultures of uterine leiomyoma cells under serum-free conditions, addition of P4 (3.18 x 10(-7) mol/L) resulted in a decrease in TNFalpha expression in the cells, relative to that in control cultures, whereas treatment with 17beta-estradiol (3.67 x 10(-8) mol/L) did not affect the TNFalpha expression in the cells. The concentrations of sex steroids used were within the physiological tissue concentrations noted in leiomyoma and myometrium. The present results suggest that the abundant expression of TNFalpha may be a molecular basis characteristic of leiomyomas in the human uterus and that P4 may play a vital role in down-regulating the expression of TNFalpha in human uterine leiomyoma.

p53 protein, a tumor suppressor gene product, has been reported to play a crucial role in suppressing the growth of a variety of cancer cells. However, little information is currently available regarding the content of p53 protein in human leiomyomas. The present study was conducted to elucidate the p53 protein content in human leiomyomas and its regulation by sex steroid hormones. The content of p53 protein in leiomyomas was examined by immunohistochemical staining and Western blot analysis in comparison with that in the adjacent normal myometrium or leiomyoma specimens from GnRH agonist-treated patients. In addition, isolated human leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% FBS for 120 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of 17 beta-estradiol (E2; 10 ng/ml), progesterone (P4; 100 ng/ml), or E2 (10 ng/ml) plus P4 (100 ng/ml). The effects of sex steroids on p53 protein content in cultured leiomyoma cells were also assessed by Western immunoblot analysis. Immunohistochemical staining and Western blot analysis revealed that p53 protein content was highest in leiomyomas treated with GnRH agonist for 16 wk, lower in leiomyomas in the secretory, P4-dominated phase, and lowest in leiomyomas in the proliferative, E2-dominated phase of the menstrual cycle. There was no difference in p53 content between leiomyomas and the adjacent normal myometrium. Western blot analysis of cultured leiomyoma cell extracts revealed that E2 treatment significantly decreased p53 protein content compared with the control cultures, whereas either P4 treatment or combined treatment with E2 and P4 did not affect p53 protein content in cultured leiomyoma cells. The concentrations of sex steroid hormones used were within the physiological tissue concentrations in leiomyomas and myometrium described earlier. The present study suggests that E2 down-regulates p53 protein content, whereas P4 is ineffective in those cells. The E2-induced decrease in p53 protein content in leiomyoma cells leads us to propose that E2 may regulate human leiomyoma growth in part by down-regulating p53 tumor suppressor protein content in those cells.

Background: Several studies show that 17β-estradiol (E2) has protective effects on atherosclerosis in the arterial wall in postmenopausal women. Little information is, however, available regarding the effect of estriol (E3) on atherosclerosis. This study was conducted to investigate the effects of E3 alone and combined E3/pravastatin therapy on intima-media thickness (IMT) of common carotid artery in postmenopausal women. Methods: Thirty-three postmenopausal women were allocated to four groups: daily treatment with E3 (2 mg) alone (E3 group, n = 10), pravastatin (10 mg) alone (pravastatin group, n = 6), combined treatment with E3 (2 mg) and pravastatin (10 mg; E3/pravastatin group, n = 7) and untreated control group (n = 10). All women attended the Kobe University Hospital once a year for routine gynecological and ultrasonographic examinations for the evaluation of atherosclerosis. Results: A significant decrease in IMT was noted in the E3/pravastatin group compared with that in the untreated control group (p < 0.05), whereas there was no significant difference in the reduction rate of IMT in the pravastatin group, E3 group and untreated control group. Conclusions: The combined E3/pravastatin therapy appeared to retard the progression of atherosclerosis in postmenopausal women.