A modest reduction in c-Myc expression has minimal effects on cell growth and apoptosis but dramatically reduces susceptibility to Ras and Raf transformation

Deregulation of c-myc and mutation of ras genes is commonly found in many h uman tumors, Several Lines of evidence indicate that c-Myc and oncogenic Ra s cooperate in causing malignant transformation, but the mechanism of this cooperation is not understood. We set out to investigate the effect on tran sformation of a modest reduction in endogenous c-Myc expression, which was achieved using a c-myc heterozygous cell line constructed by targeted homol ogous recombination. In contrast to previous reports where c-Myc expression or activity was ablated using antisense or dominant-defective methods, use of c-myc +/- cells provides a stable and homogeneous cell culture system w ith a precisely defined c-Myc expression level. In addition, this approach does not suffer from nonspecific artifacts such as antisense oligonucleotid e toxicity or interference of dominant-defective proteins with multiple (an d often undefined) target proteins. The striking and unexpected finding com municated here is that the relatively modest 50% reduction in c-Myc express ion resulted in a greater than 10-fold reduction in susceptibility to trans formation by oncogenic pas or Raf proteins. This very significant defect in transformation potential cannot be explained on the basis of a generalized cell-cycle defect, because c-myc +/- cells exhibit only a minimal (20%) re duction in proliferation. Genetic epistasis analysis indicated that c-Myc a nd Ras acted by independent pathways that converged to regulate the abundan ce of the cyclin-dependent kinase inhibitor protein p27(Kip1). Anchorage de privation elicited a strong up-regulation of p27, and a 50% reduction in c- Myc expression significantly compromised the ability of Ras to downregulate p27, We propose that Ras and c-Myc signals cooperate to regulate the activ ity of cyclin D-Cdk4/6 complexes: the former by up-regulating the expressio n of cyclin D1 and the latter by affecting the activity of the complexes. E ctopic expression of cyclin A restored the transformation potential of c-my c +/- cells, implicating it as a downstream genetic component in the pathwa y. From a therapeutic standpoint, it is of interest that, although transfor mation appears to be very sensitive to c-Myc expression levels, much Larger reductions can be tolerated without causing any significant cell cycle def ects.