c-Myc Regulates Cyclin D-Cdk4 and -Cdk6 Activity but Affects Cell Cycle Progression at Multiple Independent Points

Abstract

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 G₁and 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.

ACKNOWLEDGMENTS

This work was supported by NIH grant R01-GM-41690 to J.M.S. M.K.M. and A.J.O. were supported in part by predoctoral training grant GM-07601 from the NIH and a postdoctoral fellowship from the Ministerio Educación y Cultura de España, respectively.

We thank K. Davis for excellent technical assistance and A. Bush and M. Cole for numerous discussions and communications of unpublished observations. We gratefully acknowledge M. Ewen for GST-Rb, M. Solomon for GST-Cak1p protein, C. Sherr for p15 antibody, A. Diehl for advice on Cdk4 assays, and K. Zaret for helpful suggestions on the manuscript.

M.K.M. and A.J.O. made equal contributions to this study.