Transfection of human skeletal muscle cells with SV40 large T antigen gene coupled to a metallothionein promoter

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Abstract
We have undertaken to increase the proliferative capacity of cultured human skeletal myocytes by transfection with a plasmid construct that contains the immortalizing and transforming large T antigen gene of simian virus 40 (SV40) under the control of a zinc-sensitive metallothionein promoter. This construct was chosen to permit rapid growth of transformants in zinc-containing medium, which induces high levels of T antigen expression, and muscle-specific differentiation after withdrawal of exogenous zinc, which reduces levels of T antigen. When grown in 100 μM Zn2+, transformed myocytes expressed the large T antigen, divided rapidly, and acquired an apparently unlimited proliferative capacity. Transfer of these cells to a zinc-poor medium resulted in decreased T antigen immunofluorescence, growth rate, and saturation density as well as a return to a physiological spindle morphology. Despite transformation, these cells expressed differentiation markers characteristic of myoblasts: the B isoform of creatine kinase, and surface antigens 5.1H11, D5, and Thy 1 in the presence or absence of Zn2+. When grown to high density in a serum-poor medium, these cells differentiated further into typical multinucleated myotubes that expressed the M isoform of creatine kinase and increased levels of surface antigen 5.1H11, creatine kinase, and nicotinic acetylcholine receptors, but no detectable Thy 1 antigen. The specific activity of these differentiation markers was higher when the cells were grown in the absence of added zinc. These results indicate that transformation of human skeletal myocytes with a regulatable SV40 large T antigen gene allows an increase of the proliferative capacity of these cells with preservation of their capacity to differentiate in a physiological manner.