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Muscle Differentiation in Cell Culture

EFFECTS OF NUCLEOSIDE INHIBITORS AND ROUS SARCOMA VIRUS

From the ¹ From The Rockefeller University, New York, New York 10021

We have studied some biochemical aspects of the differentation of chick embryonic skeletal muscle cells in cell culture. Cultures are initiated in a medium that supports cell fusion and muscle cell differentiation. After 40 to 44 hours of culture, the normal onset of cell fusion is prevented by exchanging the medium for one low in Ca⁺⁺ (80 µm). After a further 24 hours, high Ca⁺⁺ (880 µm) standard medium is restored and cell fusion proceeds, followed by synthesis of creatine kinase and myokinase.

Before cell fusion is allowed to proceed, cultures are treated with 5-bromotubercidin or 5-fluorouridine, nucleoside inhibitors of RNA synthesis. 5-Fluorouridine is an irreversible inhibitor of ribosomal RNA synthesis but does not inhibit synthesis of heterogeneous nuclear RNA, or by inference, messenger RNA. 5-Bromotubercidin is a reversible inhibitor of the synthesis of both ribosomal RNA and heterogeneous nuclear RNA and of mRNA. In cultures treated with 5-fluorouridine cell fusion and synthesis of creatine kinase and myokinase proceed as in control cultures. In cultures treated briefly (8 hours) with 5-bromotubercidin, cell fusion and synthesis of creatine kinase and myokinase are delayed and lag about 16 hours behind control cultures. We conclude: (a) continuing synthesis of ribosomal RNA (or ribosomes) is not necessary for expression of differentiated muscle properties, i.e. cell fusion and enzyme synthesis. (b) After cultures are refed high Ca⁺⁺ medium (permissive for cell fusion) there must be synthesis of one or more messenger type RNA's before differentiated properties are expressed. (c) Synthesis and translation to protein of the messenger RNA(s) needed for the expression of differentiated muscle properties does not require concomitant synthesis of ribosomal RNA (or ribosomes).

Cultures of differentiating muscle cells have been infected with the RNA tumor virus Schmidt-Ruppin RSV (SR-RSV). Myotubes in infected cultures become progressively vacuolated and within 3 to 4 days after infection by SR-RSV the differentiated myotubes are destroyed. Cell fusion and synthesis of muscle enzymes continue for 2 to 3 days after cultures are infected with SR-RSV. The vacuolization and destruction of myotubes infected by SR-RSV seems to be a response analogous to the morphological transformation occurring in RSV-infected fibroblasts. The differentiated muscle cell, unlike the fibroblast, is not able to respond in a manner which would balance the deleterious effects of this tumor virus and is subsequently destroyed. Since expression of differentiated properties can begin at least 24 hours after infection by SR-RSV, it does not seem likely that transformation by SR-RSV produces an immediate effect on cell differentiation at the level of DNA.

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