MAD1 and p27^KIP1 Cooperate To Promote Terminal Differentiation of Granulocytes and To Inhibit Myc Expression and Cyclin E-CDK2 Activity

Abstract

To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27^KIP1. Generation of Mad1/p27^KIP1 double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27^KIP1, including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27^KIP1 double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27^KIP1 single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27^KIP1 operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.

We thank L. Ramos, L. Purton, S. Collins, L. Rohrschneider, G. Sale, P. Waring, and S. Tsai for technical assistance, advice, or reagents and Amir Oryan for a critical reading of the manuscript. Cytokines were generously provided by Immunex Corp., and the RXR agonist AGN 194204 was kindly provided by Allergan Corp. We are grateful to Santa Cruz Biotechnology for help with antibodies.

This work was supported by fellowships from the American Cancer Society (K.P.F.) and the Damon Runyon-Walter-Winchell Foundation DRG-076, by a Special Fellowship of the Leukemia Society of America (G.A.M.), and by grants from the National Institutes of Health (RO1CA57138 and HL54881 to R.N.E.) and from the National Health and Medical Research Council of Australia (G.A.M). R.N.E. is an American Cancer Research Professor.