Repression of G0/G1 traverse in human fibroblasts exposed to low levels of ionizing radiation

Abstract

Quiescent cultures of human fibroblasts were exposed to levels of ionizing radiation sufficient to induce a transient growth delay, while causing only small decreases in long term clonogenicity. Following the mitogenic stimulation of damaged cells, cyclin B-associated kinase activity was induced to levels equivalent to those seen in control cultures. In addition, late G 0/G1 E2F-dependent transcriptional and translational activity was observed in restimulated irradiated cells. However, cells became arrested prior to entry into S phase in a manner that paralleled the repression of cdk2-associated kinase activity. Cyclin A/cdk2-associated kinase activity was repressed in a biphasic manner following the irradiation of logarithmically growing cells. The initial rapid decline in activity to levels ∼50% of those observed in control cultures occurred prior to increases in cellular levels of p21Cip1 protein, was not blocked by the addition of cycloheximide, and was not accompanied by alterations in cdk2 phosphotyrosine content. The subsequent repression to undetectable levels was coincident with the induction of p21Cip1 and was dependent on de novo protein synthesis. Only a subpopulation of cyclin A complexes were associated with p21Cip1 regardless of the magnitude of the repression of catalytic activity, although all cyclin A-cdk2-p21Cip1 complexes were inactive. These data suggest that temporally and functionally distinct mechanisms mediate the repression of cyclin-cdk activity in damaged cells. In addition, we present evidence that irradiated cells are competent to traverse S phase and arrest in G2 in the complete absence of cdk2-associated kinase activity.