Further investigation of some inhibitors on myogenic differentiation: mechanism of inhibition with HMBA on quail myoblasts transformed with Rous sarcoma virus.

To investigate the mechanism of myogenic differentiation, we are using quail myoblast cells (QM cells) transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), termed QM-RSV cells. At 35.5 degrees C, a permissive temperature for RSV, QM-RSV cells repeatedly proliferate without differentiation, but, at 41 degrees C, a nonpermissive temperature, myogenic differentiation proceeds. This temperature dependency of the differentiation is derived from protein kinase activity of pp60v-src, as tyrosine dephosphorylation is necessary for myogenic differentiation of QM-RSV cells. In this study, it was demonstrated that among four fusion inhibitors, aspirin, doxorubicin, HMBA and TPA, three of the inhibitors, except for TPA, inhibited myogenin gene expression. Moreover, HMBA inhibited myoblast fusion accompanying inhibition of tyrosine dephosphorylation of certain proteins, and recovered the tyrosine kinase activity of pp60v-src to a certain extent. To study the effect of HMBA on the intracellular localization of pp60v-src, detergent-soluble and detergent-resistant fractions were prepared with Triton X-100. As a result, it was shown that pp60v-src mainly exists in detergent-resistant fraction at 35.5 degrees C. While almost all of the pp60v-src at 41 degrees C exists in detergent-soluble fraction. HMBA treatment retained pp60v-src in detergent-resistant fraction even at 41 degrees C. These results suggest that HMBA inhibits myogenic differentiation of QM-RSV cells by affecting the regulation of pp60v-src.