Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G 2-phase arrest and subsequent G 1-phase arrest: TP53-dependent and TP53-independent roles

Abstract

Through a detailed study of cell cycle progression, protein expression, and kinase activity in γ-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G 2-phase and subsequent G 1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G 2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G 2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G 2 phase, normal cells chronically arrest in the subsequent G 1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G 1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21 WAFI/CIPI) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G 1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G 1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity. © 2003 by Radiation Research Society.