Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in human uterine leiomyoma cells.
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.
Gao
Z
Z
Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Matsuo
H
H
Wang
Y
Y
Nakago
S
S
Maruo
T
T
D000328:Adult; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007889:Leiomyoma / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects*; D014594:Uterine Neoplasms / Q000235:genetics / Q000378:metabolism*
D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D007334:Insulin-Like Growth Factor I