Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in human uterine leiomyoma cells.

Abstract

IGF-I has been reported to play a role in regulating proliferation of human leiomyoma cells. There is, however, little evidence to suggest that IGF-I inhibits apoptosis in the leiomyoma cells. The present study was conducted to elucidate whether IGF-I affects apoptosis and Bcl-2 protein expression, an apoptosis-inhibiting gene product, in cultured leiomyoma cells. In addition, we examined the effect of IGF-I on proliferating cell nuclear antigen (PCNA) expression in cultured leiomyoma cells. 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 graded concentrations of IGF-I (1.0, 10, and 100 ng/ml). The effects of IGF-I on Bcl-2 protein and PCNA expression in cultured leiomyoma cells were assessed by Western immunoblot analysis and immunocytochemical staining, whereas the effects of IGF-I on the cell viability and apoptosis of the cultured cells were determined by 3-(4,5-dimethylatriazol-2-yl)-2,5diphenyltetrasodium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay, respectively. Immunocytochemical staining demonstrated that IGF-I treatment resulted in the increase in PCNA labeling index in cultured leiomyoma cells in a dose-dependent manner. Immunoblot analysis of proteins extracted from the cultured leiomyoma cells revealed that the addition of IGF-I (10 and 100 ng/ml) significantly increased the expression of 35-kDa immunoreactive PCNA and 26-kDa Bcl-2 protein, compared with those in control cultures. Cell survival and proliferation of cultured leiomyoma cells, assessed by MTT assay, was significantly augmented by IGF-I treatment, compared with those of control cultures. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay showed that the apoptosis-positive rate of leiomyoma cells treated with IGF-I was significantly decreased, compared with that in control cultures. The present results suggest that IGF-I plays crucial roles in leiomyoma cell growth, not only in promoting the proliferative potential by up-regulation of PCNA expression but also in down-regulating apoptosis by up-regulation of Bcl-2 protein expression in leiomyoma cells.