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

Dergulation of c-myc and mutation of ras genes is commonly found in many human tumors. Several lines of evidence indicate that c-Myc and oncogenic Ras cooperate in causing malignant transformation, but the mechanism of this cooperation is not understood. We set out to investigate the effect on transformation of a modest reduction in endogenous c-Myc expression, which was achieved using a c-myc heterozygous cell line constructed by targeted homologous 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 with a precisely defined c-Myc expression level. In addition, this approach does not suffer from nonspecific artifacts such as antisense oligonucleotide toxicity or interference of dominant-defective proteins with multiple (and often undefined) target proteins. The striking and unexpected finding communicated here is that the relatively modest 50% reduction in c-Myc expression resulted in a greater than 10-fold reduction in susceptibility to transformation by oncogenic Ras 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%) reduction in proliferation. Genetic epistasis analysis indicated that c-Myc and Ras acted by independent pathways that converged to regulate the abundance of the cyclin-dependent kinase inhibitor protein p27Kip1. Anchorage deprivation elicited a strong up-regulation of p27, and a 50% reduction in c-Myc expression significantly compromised the ability of Ras to down-regulate p27. We propose that Ras and c-Myc signals cooperate to regulate the activity of cyclin D-Cdk4/6 complexes: the former by up-regulating the expression of cyclin D1 and the latter by affecting the activity of the complexes. Ectopic expression of cyclin A restored the transformation potential of c-myc +/- cells, implicating it as a downstream genetic component in the pathway. From a therapeutic standpoint, it is of interest that, although transformation appears to be very sensitive to c-Myc expression levels, much larger reductions can be tolerated without causing any significant cell cycle defects.

Identification of CDK4 as a target of c-MYC.

The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.

Mysterious liaisons: the relationship between c-Myc and the cell cycle.

A large body of physiological evidence shows that either upregulation or downregulation of intracellular c-Myc activity has profound consequences on cell cycle progression. Recent work suggests that c-Myc may stimulate the activity of cyclin E/cyclin-dependent kinase 2 (Cdk2) complexes and antagonize the action of the Cdk inhibitor p27KIP1. Cyclin D/Cdk4/6 complexes have also been implicated as targets of c-Myc activity. However, in spite of considerable effort, the mechanisms by which c-Myc interacts with the intrinsic cyclin/Cdk cell cycle machinery remain undefined.

c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points.

c-myc is a cellular proto-oncogene associated with a variety of human cancers and is strongly implicated in the control of cellular proliferation, programmed cell death, and differentiation. We have previously reported the first isolation of a c-myc-null cell line. Loss of c-Myc causes a profound growth defect manifested by the lengthening of both the G1 and G2 phases of the cell cycle. To gain a clearer understanding of the role of c-Myc in cellular proliferation, we have performed a comprehensive analysis of the components that regulate cell cycle progression. The largest defect observed in c-myc-/- cells is a 12-fold reduction in the activity of cyclin D1-Cdk4 and -Cdk6 complexes during the G0-to-S transition. Downstream events, such as activation of cyclin E-Cdk2 and cyclin A-Cdk2 complexes, are delayed and reduced in magnitude. However, it is clear that c-Myc affects the cell cycle at multiple independent points, because restoration of the Cdk4 and -6 defect does not significantly increase growth rate. In exponentially cycling cells the absence of c-Myc reduces coordinately the activities of all cyclin-cyclin-dependent kinase complexes. An analysis of cyclin-dependent kinase complex regulators revealed increased expression of p27(KIP1) and decreased expression of Cdk7 in c-myc-/- cells. We propose that c-Myc functions as a crucial link in the coordinate adjustment of growth rate to environmental conditions.

c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets.

We report here that the expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. Our results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes.

Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination.

Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +/+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-->S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.