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.
