Exogenous expression of Msx1 renders myoblasts refractory to differentiation into myotubes and elicits enhanced biosynthesis of four unique mRNAs

Murine myoblast cell lines stably transfected with expression vectors conta ining homeobox Msx1 cDNA in sense (F31-c) or antisense (F3R1) orientation h ave contrasting phenotypes. F3R1 cells readily differentiate in medium cont aining low serum whereas F31-c cells fail to differentiate under these cond itions. The mechanism by which exogenous overexpression of Msx1 leads to th e altered phenotype of F31-c cells and the downstream targets of Msx1 are u nknown. Using the method of differential display, we have identified four c DNAs that represent transcripts up-regulated in F31-c. Two of these cDNAs a re homologous to ribosomal proteins S23 and S24 while the third has homolog y to sequences in the murine Tcp-1 gene. A fourth cDNA does not have apprec iable homology to cDNA sequences deposited in the NIH GenBank. Since withdr awal from the cell cycle and enhanced expression of MyoD commonly precede d ifferentiation of myoblasts into myotubes, we also examined regulation of t he major cell cycle proteins as well as MyoD by Western blot analysis. We s how that the levels of Cdks 2, 4 and 6, cyclins A and D, and the Cdk inhibi tor p27 in both proliferating and serum-starved F31-c cells were similar to those in F3R1. Finally, although MyoD protein levels increased in both cel l types after 72 h incubation in serum depleted medium, the levels of MyoD in serum-starved F31-c cells were 2-4 fold lower. We postulate that the red uced amount of MyoD is sufficient to permit reversible withdrawal of F31-c cells from the cell cycle, but is inadequate to permit myogenesis.