The biochemical mechanisms responsible for antitumor drug- and radiation- induced G2 arrest and cell death in mammalian cells are poorly understood. Preliminary data indicate that cdc2 kinase activity, which is required for mammalian cells to enter mitosis, is rapidly inhibited upon treatment of Chinese hamster ovary (CHO) cells with etoposide or gamma-radiation. The major aim of this proposal is to further define the role that mammalian cdc2 may play in G2 arrest and cell death. In order for this to be achieved, a broad spectrum of antitumor agents will be employed to determine their effects on cell cycle progression and cdc2 kinase activity. These effects will be related to formation of specific DNA lesions (single- and double-strand breaks, DNA-DNA crosslinks, and DNA-protein crosslinks), as measure by neutral- and alkaline-elution. The mechanism of cdc2 kinase inhibition in CHO cells will be studied. This will include detailed analyses of the turnover of cdc2 itself, or of proteins which may interact with cdc2 and regulate its activity. In addition, alterations in the phosphorylation state of cdc2 will be studied following drug treatment or irradiation in order to determine whether these may account for enzyme inhibition. Biochemical modulators of G2 arrest will be utilized, not only to define the role of cdc2 in G2 arrest, but also to facilitate characterization of drug- and radiation-induced cell death with respect to cdc2 kinase activity. Further understanding of the role of cdc2 in G2 arrest and cell death may be obtained by selection and characterization of hypersensitive CHO mutants which lack the ability to arrest in G2 following treatment with drugs or radiation. The possibility will also be explored that different mechanisms of cytotoxicity are induced in different human tumors treated with the same antitumor agent. Finally, cdc2 peptide polyclonal antisera will be raised and characterized. The experiments described herein are important, not only in defining mechanisms of action of antitumor agents, but also in identifying novel targets for therapeutic exploitation.
