P53 mutations are found in more than 50% of human cancers. It is known that expression of the temperature sensitive p53 missense mutation 135Val inhibits differentiation of myogenic C2 cells, which express wild type p53, but does not affect their ability to exit the cell cycle (S Soddu et al.). In vivo, however, p53 is not required for myogenesis (LA Donehower et al.; T Jacks et al.). The proposed experiments test two hypotheses. The first is that wild type (wt) p53 possesses an activity that contributes to the progress of myogenesis but is not required for withdrawal from the cell cycle. The second is that different mutant forms of p53 possess different abilities to inhibit the progress of myogenesis, either by differential inactivation of various p53 activities, or by a gain of function, independent of the ability to functionally inactivate wt p53. These hypotheses will be addressed by using stable and transient transfections in cells which are null for p53 or in which p53 has been inactivated, either by anti-sense, the Adenoviral E1B-55 kD protein, or a dominant negative p53 mutation. Th effects of p53 missense and denatured conformations of dominant negative p53, will be compared. Resulting phenotypes will be evaluated by microscopy for myotube formation. Identification of phenotypic differences between derivative cell lines and the control lines will provide clues as to the mechanism(s) by which p53 acts. Immunoblot analyses will e used to assess the accumulation and modification of several proteins that have been shown to be important in the progress of myogenesis. Difference libraries will be constructed to facilitate identification of transcriptional events affected by p53. Reporter assays will assess the effects of wt and mutant p53s on promoters activated by myogenesis regulatory and enhancing factors (MRFs and MEF2s). Rescue experiments will be used to determine what molecules other than wt p53 restore the normal phenotype. Knowledge of why a mutant p53 inhibits myogenesis in culture will increase our knowledge both of the activities by which p53 controls growth and differentiation and out of the steps in myogenesis.
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