TRANSFORMING GROWTH FACTOR (TGF)-BETA IN RHABDOMYOSARCOMA

TGF-β 1在正常的肌生成中具有抑制作用。 也造成 可逆抑制肌肉特异性基因的融合和表达 骨骼肌和平滑肌成肌细胞体外培养 增殖因此，有人推测TGF-β 1可能具有 在肌肉再生中的作用，并可能防止过早融合 胚胎成肌细胞 TGF-β在肌源性肿瘤中的作用尚不清楚。 用抗生物素蛋白-生物素免疫过氧化物酶技术，我们发现， 横纹肌肉瘤（RMS）对TGF-β 1和TGF-β 3染色强烈，但 而不是体内的TGF-β 2。 该项目旨在研究可能的 TGF-β在RMS体外生长和分化中的作用。 三 选择已建立的RMS细胞系（RD、Birch和RH 18）用于 问题研究 用3 H-胸腺嘧啶核苷掺入法，我们发现TGF-β 1， 0.25 - 1 ng/ml浓度的β 1抑制所有3种细胞的生长 在无血清培养基中生长的RMS细胞系。 这种抑制作用出现在 特异性针对外源性而非内源性TGF-β 1， 抗体实验 由于TGF-β 1 mRNA在所有3种RMS细胞中均被检测到， 其中2株在TGF-β 1处理后增加， 提示TGF-β 1的自分泌作用， 内源性TGF-β 1功能可能是由于内源性TGF-β 1的非活性形式， TGF-β 1，这通常是体外肿瘤模型的情况。 澄清 这个问题，TGF-β 1蛋白水平将评估条件 来自所有3种细胞系的培养基，其中含有貂肺成纤维细胞（CCL-64） 生物测定 TGF-β 1对细胞增殖的影响将是 结合细胞周期mRNA水平的变化进行分析 相关基因，如c-myc，c-fos和c-jun。 通过形态学（细胞融合试验）以及 肌肉决定基因mRNA水平的可能变化， MyoD 1和Myogenin以及肌肉蛋白基因结蛋白和CK-MM。肌肉 蛋白质表达将通过免疫荧光进行评价， 市售抗体。

TGF-beta1 has an inhibitory effect in normal myogenesis. It also causes reversible inhibition of fusion and expression of muscle specific genes by skeletal and smooth muscle myoblasts in vitro without affecting cell proliferation. Therefore, it has been postulated that TGF-beta1 may have a role in muscle regeneration and may prevent precocious fusion of embryonic myoblasts. The role of TGF-beta in myogenous tumors is unknown. Using the avidin-biotin immunoperoxidase technique, we found that rhabdomyosarcomas (RMS) stain intensely for TGF-beta1 and TGF-beta3, but not TGF-beta2 in vivo. This project aims at investigating the possible role of TGF-beta in the growth and differentiation of RMS in vitro. Three established RMS cell lines (RD, Birch and RH18) are selected for the studies. Using the 3H-thymidine incorporation assay, we found that TGF- beta1 at concentrations of 0.25 to 1 ng/ml inhibits cell growth in all 3 RMS cell lines grown in serum-free media. This inhibitory effect appeared to be specific to exogenous, but not endogenous TGF-beta1 with blocking antibody experiments. Since TGF-beta1 mRNA was detected in all 3 RMS cell lines and increased after treatment with TGF-beta1 in 2 of them, suggesting an autocrine role for TGF-beta1, lack of detection of endogenous TGF-beta1 function may be due to an inactive form of endogenous TGF-beta1, which is usually the case in tumor models in vitro. To clarify this issue, TGF-beta1 protein levels will be evaluated in conditioned media from all 3 cell lines with the mink lung fibroblast (CCL-64) bioassay. The effects of TGF-beta1 on the cell proliferation will be analyzed in conjunction with changes concerning mRNA levels of cell cycle related genes, such as c-myc, c-fos and c-jun. Differentiation will be evaluated by morphology (cell fusion assay), as well as detection of possible changes in the mRNA levels of the muscle determination genes, MyoD1 and Myogenin and the muscle protein genes desmin and CK-MM. Muscle protein expression will be evaluated by immunofluorescence using commercially available antibodies.