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.
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