THE ROLE OF TRANSFORMING GROWTH FACTOR BETA IN RHABDOMYOSARCOMA

我们发现，横纹肌肉瘤显示一贯高水平的TGF-β 1， TGF-β 1，和在较小程度上TGF-β 3，但缺乏TGF-β 2蛋白， 免疫组织化学染色。 我们还检测到不同水平的TGF- 横纹肌肉瘤条件培养液中β蛋白的合成 (RMS)使用基于TGF-β抑制DNA的生物测定法 在水貂肺成纤维细胞（CCL-64细胞）中的合成。 此外，所有横纹肌肉瘤表达不同水平的TGF-β 1 mRNA， 体外 这些数据，结合我们以前的TGF-β- 诱导抑制RMS细胞分化的体外，和已知的 TGF-β在正常肌生成中抑制作用已经提示我们 TGF-β在人RMS中可能的自分泌抑制作用。 这 本研究将从以下几个方面对这一假说进行研究：（1）RMS细胞系 表达高水平和低水平TGF-β mRNA将被研究， 通过用以下物质孵育的细胞裂解物的PAGE检测TGF-β受体的存在 （125）I-TGF-β 1、2、3。 (2)抗TGF-β阻断抗体和 将使用反义寡核苷酸研究干扰 细胞生长（通过3H-胸苷掺入进行DNA合成）， 肌原性分化的抑制（肌管融合测定），在 存在或不存在浓度增加的外源性TGF-β。 由于TGF-β受体在成肌细胞融合成肌细胞后减少， 肌管，我们以前的数据已经显示了TGF-β的更有效的作用， 在具有更原始形态的细胞系中，我们还将 评估融合阻滞（与 地西泮或EGTA）与5-氮杂胞苷分化的RMS细胞相比。 也将评价视黄酸（RA）处理的细胞，因为RA是 发现在某些细胞类型中诱导TGF-β受体表达。

We found that rhabdomyosarcomas show consistently high levels of TGF- beta1, and to a lesser extent TGF-beta3, but lack TGF-beta2 protein by immunohistochemical staining. We also detected variable levels of TGF- beta protein synthesis in conditioned media of cultured rhabdomyosarcoma (RMS) cells using a bioassay based on TGF-beta inhibition of DNA synthesis in mink lung fibroblasts (CCL-64 cells). Furthermore, all RMS expressed variable levels of TGF-beta1 mRNA in vitro. These data, in combination with our previous data of TGF-beta- induced inhibition of RMS cell differentiation in vitro, and the known inhibitory effect of TGF-beta in normal myogenesis, have suggested to us a possible autocrine inhibitory role of TGF-beta in human RMS. This hypothesis will be studied in the following ways: (1) RMS cell lines expressing high and low levels of TGF-beta mRNA will be studied for the presence of TGF-beta receptors by PAGE of cell lysates incubated with (125)I-TGF-beta 1,2,3. (2) Anti-TGF-beta blocking antibodies and antisense oligonucleotides will be employed to investigate interference with cell growth (DNA synthesis by 3H-thymidine incorporation) and inhibition of myogenic differentiation (myotube fusion assay), in the presence or absence of increased concentration of exogenous TGF-beta. Since TGF-beta receptors decrease after fusion of myoblasts into myotubes, and our previous data have shown a more potent action of TGF- beta in cell lines with a more primitive morphology, we will also evaluate levels of TGF-beta mRNA and protein in fusion arrested (with diazepam or EGTA) , versus 5-azacytidine differentiated RMS cells. Retinoic acid (RA)-treated cells will be evaluated as well, since RA was found to induce TGF-beta receptor expression in some cell types.