Effects of transforming growth factor beta 1 on the growth of rhabdomyosarcoma cell line RD

　　转化生长因子对 横纹肌肉瘤细胞系 RD的影响

　　Abstract：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 cyclin E-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.

　　摘要：转化生长因子(TGF-β)能抑制多数上皮细胞和内皮细胞生长。TGF-β的增长调节作用在软组织肉瘤中很少报道。现在我们检测TGF-β1 影响人类横纹肌肉瘤细胞系RD和调查相关的分子机制。方法：用[3H]胸腺嘧啶及(MTT)化验检测 RD分裂能力。流式细胞术分析RD细胞周期。 RD细胞周期的蛋白质和信使RNA分别用蛋白质印迹和逆转录聚合酶链反应(RT-PCR)检测。cdk2 or cdk4的活性用免疫沉淀反应和激酶的测定。免疫荧光染色用于检测细胞周期的调节因子在RD的位置，结果：TGF-β1抑制RD 增殖通过G1-逮捕在细胞周期进程。TGF-β1能促进 RD的P27，然P27增多能绑定到细胞周期蛋白E-cdk2 形成复合体，从而有效地抑制cdk2激酶活性。P21增加和RD原癌基因减少是因为 TGF-β1。P15 and cdk4没有参与生长抑制。TGF-β1治疗诱导P27聚集于细胞核。通过TGF-β1治疗P27从细胞核弥漫到细胞核。结论：TGF-β1人类的横纹肌肉瘤细胞系RD 增殖和诱发RD G1-逮捕。这个过程是通过TGF-β1升高 P27去抑制cdk2激酶活性。P21的感应和 C-myc下降可以参与增长-逮捕事件