Transfection of human skeletal muscle cells with SV40 large T antigen gene coupled to a metallothionein promoter.
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 microM 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.
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D002038:Bungarotoxins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D006801:Humans; D008668:Metallothionein / Q000235:genetics*; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011978:Receptors, Nicotinic / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D002038:Bungarotoxins; D009856:Oncogene Proteins, Viral; D011978:Receptors, Nicotinic; D008668:Metallothionein; D003402:Creatine Kinase; D015032:Zinc