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Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorticoid and DNA damage but not in CD95-induced apoptosis in thymocytes. Apoptotic Cdk2 activation in lymphoid cells is controlled by a recently identified protein, cyclin O, and its activity is modulated by p53 and members of the Bcl-2 protein family. In this chapter, we describe methods for measuring changes in Cdk2 activity during apoptosis. In addition, we also show the details of the generation of an antibody able to immunoprecipitate the cyclin O complexes from apoptotic cells in native conditions and its use to measure the kinase activity associated with this proapoptotic cyclin.

Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for the apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorticoid and DNA damage-induced but not in CD95-induced apoptosis in thymocytes. Apoptotic Cdk2 activation is controlled by apoptosis regulatory proteins like p53 and Bcl2 family members and correlates with degradation of its inhibitors p21Cip1 and p27Kip1 by activated caspases. Methods for measuring Cdk2 changes in activity in quiescent cells undergoing apoptosis are detailed in this chapter.