It is notewor thy that induction of myogenic differentiation in MadMyc chimera expressing cells does not imply a myogenin or MyoD increased expression level neither down regulation of pospho ERKs MEK162 ARRY-162 which are instead enhanced. This is in agreement with the role of ERKs in fusion and late differ entiation processes during myogenic differentiation. Importantly, MadMyc stably expressing cells do not exhibit anchorage independent growth, which is instead enhanced in c Myc over expressing cells. On the other hand, forced expression of c Myc attenuated the U0126 mediated anchorage independent growth inhibition and differentiative effects in RD cells. These experiments dem onstrate that c Myc over expression rescues oncogenic phenotype repressed by MEK inhibitor U0126.
Worthy of note is also the fact that the role of mutated Ras in aber rant growth of RD cells is compromised by the selective disruption of c Myc in MadMyc expressing cells demon Inhibitors,Modulators,Libraries strating that c Myc is indispensable to the maintaining of Ras/MEK/ERK mediated oncogenic phenotype. Conclusion Our data provide evidence that the cooperation between MEK/ERK and c Myc pathways play a major role in the expression of transformed phenotype in muscle and non muscle derived transformed cell lines. Importantly, our results show for the first time that the disruption of c Myc pathway either directly or indirectly drammatically impairs the expression of transformed phenotype induc ing myogenic differentiation in RD cells. In conclusion these data strongly suggest that the targeting of c Myc by means of the MEK/ERK inhibitor can be tested as a prom ising strategy in anti cancer therapy.
Inhibitors,Modulators,Libraries Methods Cell cultures and treatments The embryonal Rhabdomyosarcoma, the prostate carcinoma PC3, the melanoma IGR39 and colon adenocarcinoma SW403 human cancer cell lines were cultured in Dulbecco modified Eagle medium, supple mented with glutamine, gentamycin and 10% or 15% heat inactivated foetal Inhibitors,Modulators,Libraries bovine serum. C2C12 and NIH3T3 were grown in DMEM supplemented with glutamine, gentamycin and 10% Inhibitors,Modulators,Libraries heat inactivated foetal bovine serum. One day after plating, cells were treated with 10M U0126 kinase Inhibitors,Modulators,Libraries inhibitors or 10 7 M TPA for the times shown in the figures. Immunoprecipitation Cells were harvested in phosphate buffered saline, sedi mented and lysed in 10 mM Tris pH 7, 50 mM NaCl, 1% NP40, 1 mM ZnCl2, additioned with protease and phos phatase inhibitors. Protein extracts were clarified by Tubacin mw cen trifugation. Supernatant, normalized as equal amounts of proteins, were incubated with Max antibody at 4 C for 3 hrs. 30l of protein G Plus were added to collect immunocomplexes. Protein G bound immunocomplexes were washed 6 times with extraction buffer and processed for SDS PAGE and immunoblotting.