Development of ‘revertant’ myotubes in cultures of Rous sarcoma virus transformed avian myogenic cells

Abstract

Embryonic chick and quail skeletal myogenic cells were infected with several temperature-sensitive mutants of Rous sarcoma virus ( tsRSV). Cultures were passaged at least four times at permissive temperature (35° C) to ensure transformation of the entire population. When maintained at 35° C, the transformed myogenic cells were indistinguishable from transformed fibroblasts. However, upon subculturing at nonpermissive temperature (41° C) many of the infected cells lost their transformed phenotype and withdrew irreversibly from the cell cycle. Many developed into mononucleated myoblasts, whereas others fused into typical elongated, smooth contoured, multinucleated myotubes. These postmitotic myoblasts and myotubes initiated the synthesis of muscle-specific myosin and assembled these molecules into typical striated myofibrils. They also initiated the synthesis of desmin, the muscle-specific intermediate-sized 10 nm filaments, acetylcholine receptors (AChR), and the MM-isozyme of creatine-phosphokinase (MM-CPK). Upon subculturing at permissive temperature the great majority of cells continued to replicate and retained their transformed phenotype. Nevertheless, even at permissive temperature a small number of transformed cells ‘spontaneously’ withdrew from the cell cycle and formed morphologically atypical postmitotic myoblasts and/or myotubes. These atypical postmitotic myotubes were exceedingly flat with irregular contours and centrally clustered nuclei. The muscle-specific myosin synthesized in these cells was largely confined to a diffuse perinuclear zone and the desmin filaments exhibited an intracellular distribution characteristic of immature myogenic cells. By altering culture conditions, the number of myotubes formed at permissive temperature could be greatly augmented. This was correlated positively with higher levels of AChR and MMCPK. Clearly, the degree of suppression of the differentiation program of myogenic cells by RSV can be modulated by environmental conditions.