Backgroud Transforming growth factor-β (TGF-β) can inhibit the growth of most epithelial and endothelial cells. The growth regulative role of TGF-β on soft tissue sarcoma was seldom reported. Here we examined TGF-...Backgroud Transforming growth factor-β (TGF-β) can inhibit the growth of most epithelial and endothelial cells. The growth regulative role of TGF-β on soft tissue sarcoma was seldom reported. Here we examined TGF-β1 effects on the growth of human rhabdomyosarcoma cell RD and searched the relative molecular mechanism. Methods The viability of RD was examined by [ 3H]-thymidine incorporation and [3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetraz olium bromide] (MTT) assay. RD cell cycle was analysed by flow cytometry. The protein and mRNA of cell cycle regulative factors in RD were detected by Western blot and reverse transcription-polymerase chain reation (RT-PCR), respectively. The kinase activity of cdk2 or cdk4 was examined by immunoprecipitation and kinase assay. Immunofluorescent staining was used to detect the location of cell cycle regulative factors in RD by laser scanning confocal microscope.Results TGF-β1 inhibits RD proliferation by G1-arrest in cell cycle progression. TGF-β1 can prominently up-regulate P27 of RD, then augment P27 to bind cyclinE-cdk2 complexes, which effectively suppress cdk2 kinase activity. P21 increased and c-myc decreased in RD due to TGF-β1. Both P15 and cdk4 have not been involved in the growth inhibitory event. TGF-β1 treatment induced P27 to congregate around nucleus. P21 pervaded from nucleus to both nucleus and cytoplasm by TGF-β1 treatment.Conclusion TGF-β1 inhibits the proliferation of human rhabdomyosarcoma cell line RD and induces RD G1-arrest. This course is accomplished by TGF-β1 up-regulating P27 to suppress cdk2 kinase activity. The induction of P21 and down-regulation of C-myc might participate in the growth-arrest event.展开更多
Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescen...Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescence, slot-blot and dye-transfer methods. These cells grow rapidly and show aberrant and incomplete myogenic differentiation. To investigate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD single-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slowly and displays better myogenic differentiation. The expression of Cx43, its mRNA and the GJIC in RDL3 is comparable to that in normal myoblasts. Another subclone RDL6 which grows rapidly, but is poorly differentiated, expresses very low levels of Cx43 and its mRNA, and very weak GJIC. By using the calcium phosphate precipitate transfection technique, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduced into the RDL6 cells. Several stably transfected clones have been obtained. A stable Cx43-transfectant clone RDL6/C4 expresses high level of Cx43 and its mRNA, and results in dramatic increase of GJIC. These cells grow slowly but display the enhanced myogenic differentiation. A correlation between the down-regulation of Cx43 gene expression and a reduced expression of myogenic differentiation in RD cells is demonstrated. Forced expression of Cx43 not only inhibits cell growth but also correlates with the improved myogenic differentiation of RD cells.展开更多
文摘Backgroud Transforming growth factor-β (TGF-β) can inhibit the growth of most epithelial and endothelial cells. The growth regulative role of TGF-β on soft tissue sarcoma was seldom reported. Here we examined TGF-β1 effects on the growth of human rhabdomyosarcoma cell RD and searched the relative molecular mechanism. Methods The viability of RD was examined by [ 3H]-thymidine incorporation and [3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetraz olium bromide] (MTT) assay. RD cell cycle was analysed by flow cytometry. The protein and mRNA of cell cycle regulative factors in RD were detected by Western blot and reverse transcription-polymerase chain reation (RT-PCR), respectively. The kinase activity of cdk2 or cdk4 was examined by immunoprecipitation and kinase assay. Immunofluorescent staining was used to detect the location of cell cycle regulative factors in RD by laser scanning confocal microscope.Results TGF-β1 inhibits RD proliferation by G1-arrest in cell cycle progression. TGF-β1 can prominently up-regulate P27 of RD, then augment P27 to bind cyclinE-cdk2 complexes, which effectively suppress cdk2 kinase activity. P21 increased and c-myc decreased in RD due to TGF-β1. Both P15 and cdk4 have not been involved in the growth inhibitory event. TGF-β1 treatment induced P27 to congregate around nucleus. P21 pervaded from nucleus to both nucleus and cytoplasm by TGF-β1 treatment.Conclusion TGF-β1 inhibits the proliferation of human rhabdomyosarcoma cell line RD and induces RD G1-arrest. This course is accomplished by TGF-β1 up-regulating P27 to suppress cdk2 kinase activity. The induction of P21 and down-regulation of C-myc might participate in the growth-arrest event.
基金Project supported by the National Natural Science Foundation of China and the exchange program between China and Canada.
文摘Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescence, slot-blot and dye-transfer methods. These cells grow rapidly and show aberrant and incomplete myogenic differentiation. To investigate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD single-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slowly and displays better myogenic differentiation. The expression of Cx43, its mRNA and the GJIC in RDL3 is comparable to that in normal myoblasts. Another subclone RDL6 which grows rapidly, but is poorly differentiated, expresses very low levels of Cx43 and its mRNA, and very weak GJIC. By using the calcium phosphate precipitate transfection technique, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduced into the RDL6 cells. Several stably transfected clones have been obtained. A stable Cx43-transfectant clone RDL6/C4 expresses high level of Cx43 and its mRNA, and results in dramatic increase of GJIC. These cells grow slowly but display the enhanced myogenic differentiation. A correlation between the down-regulation of Cx43 gene expression and a reduced expression of myogenic differentiation in RD cells is demonstrated. Forced expression of Cx43 not only inhibits cell growth but also correlates with the improved myogenic differentiation of RD cells.
