Inhibition of DNA synthesis and G1/S-phase transition in normal human fibroblasts elicited by a heat-labile trans-acting factor in gamma-irradiated HeLa cell extracts.

Proliferating human cells exposed to ionizing radiation show complex cellular responses including a delay in progression through various phases in the cell cycle. These cell cycle checkpoints are regulated by mitogenic signaling pathways which transduce the extracellular signals to the cell cycle control machinery. In this study we demonstrate that microinjection of a cellular extract, prepared from gamma-irradiated (40 Gy) HeLa cells, into the cytoplasm of normal human fibroblasts results in suppression of DNA replicative synthesis, indicating the presence of a trans-acting DNA synthesis-inhibiting factor(s). The addition of this same extract to the culture medium for a short time (< or = 2 h) also inhibits DNA synthesis in human fibroblasts, affecting both replicon initiation and DNA chain elongation processes. Moreover, a 2-h incubation of the fibroblast cultures with the extract causes a transient delay in cell progression from G1 to S phase coupled with up-regulation of the p53 tumor suppressor protein. Both the DNA synthesis-inhibiting and G1-phase-blocking activities are reduced markedly when the extract is heated (80 degrees C; 10 min) prior to its addition to the culture medium. On the other hand, pretreatment of the fibroblast cultures with KN62, an inhibitor of calmodulin-dependent kinase II (CaMKII), serves to abrogate the inhibitory effect of the extract on DNA synthesis without influencing its ability to induce the G1-phase block. These results are compatible with the presence in HeLa cell extracts of a heat-labile trans-acting factor that triggers, in normal human cells, the activation of (1) a CaMKII-dependent signal transduction pathway mediating suppression of DNA synthesis and (2) a p53-dependent pathway mediating G1-phase checkpoint control.