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A modest reduction in c-myc expression has minimal effects on cell growth and apoptosis but dramatically reduces
A V Bazarov1, S Adachi, S F Li
1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
Abstract:
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.
Insights
A 50% reduction in c-Myc expression significantly impairs tumor transformation driven by Ras and Raf. This occurs independently of cell cycle defects, highlighting c-Myc
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Deregulation of c-Myc and mutations in Ras genes are common in human tumors.
- c-Myc and oncogenic Ras are known to cooperate in malignant transformation, but the underlying mechanism remains unclear.
- Previous studies on c-Myc's role in transformation used methods with potential artifacts.
Purpose of the Study:
- To investigate the effect of a modest reduction in endogenous c-Myc expression on cellular transformation.
- To elucidate the mechanism by which c-Myc and Ras cooperate in transformation.
- To establish a stable and artifact-free system for studying c-Myc's role in transformation.
Main Methods:
- Construction and utilization of a c-myc heterozygous (c-myc +/-) cell line via targeted homologous recombination.
- Assessment of transformation susceptibility upon oncogenic Ras or Raf protein expression.
- Genetic epistasis analysis to determine pathway interactions and examination of p27Kip1 regulation.
- Investigation of cyclin D1, cyclin A, and cyclin D-Cdk4/6 complex activity.
Main Results:
- A 50% reduction in c-Myc expression led to a >10-fold decrease in transformation susceptibility by Ras or Raf.
- This defect in transformation was not due to generalized cell-cycle defects, as proliferation was only minimally affected.
- c-Myc and Ras act via independent pathways converging on p27Kip1 regulation; c-Myc reduction impaired Ras-mediated p27 downregulation.
- Ectopic expression of cyclin A rescued the transformation potential of c-myc +/- cells.
Conclusions:
- Modest reduction in c-Myc significantly impairs oncogenic Ras/Raf-driven transformation, independent of major cell cycle defects.
- c-Myc and Ras cooperate by regulating p27Kip1 levels, likely through modulation of cyclin D-Cdk4/6 activity.
- Cyclin A is a downstream effector in the cooperative transformation pathway.
- Therapeutic strategies targeting c-Myc levels may be effective, as transformation is sensitive to its expression, while significant reductions are tolerated by normal cells.