INTERMEDIATE FILAMENT PROTEINS IN TPA-TREATED SKELETAL-MUSCLE CELLS IN CULTURE

The cocarcinogenic phorbol ester 13-tetradecanoyl-O-phorbol acetate se lectively and reversibly inhibits the ongoing differentiation programm e of chick muscle cells in culture. 13-tetradecanoyl-O-phorbol acetate promptly blocks spontaneous contractions in mature myotubes and induc es them to retract, forming giant myosacs and concurrently stress fibr e-like structures are assembled. Using indirect immunofluorescence to localise desmin, the muscle specific intermediate filament protein, it was shown that its distribution is longitudinally oriented in mature myotubes. In myosacs, desmin has a reticular pattern although not as l inearly oriented as in control myotubes. Using gel electrophoresis of control and 13-tetradecanoyl-O-phorbol acetate treated cell extracts, three major protein bands were observed with molecular weight of 43, 5 0 and 55 kDa. They migrate as actin, desmin and vimentin, respectively . The 50 kDa and 55 kDa proteins were expressed more in 13-tetradecano yl-O-phorbol acetate-treated cells. The 50 kDa band was confirmed as d esmin by immunoblotting using anti-chicken desmin antibody. Two-dimens ional gel electrophoresis analysis showed the appearance of more acidi c isoforms of the 50 and 55 kDa proteins 13-tetradecanoyl-O-phorbol in acetate-treated cells. The 43 kDa protein was seen as three distinct isoforms in control cells and as only two isoforms in 13-tetradecanoyl -O-phorbol acetate-treated cells.