INHIBITION OF DNA-SYNTHESIS AND G(1) 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 c ellular responses including a delay in progression through various pha ses in the cell cycle. These cell cycle checkpoints are regulated by m itogenic 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 res ults in suppression of DNA replicative synthesis, indicating the prese nce of a trans-acting DNA synthesis-inhibiting factor(s). The addition of this same extract to the culture medium for a short time (less tha n or equal to 2 h) also inhibits DNA synthesis in human fibroblasts, a ffecting 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 G(1) to S phase cou pled with up-regulation of the p53 tumor suppressor protein. Both the DNA synthesis-inhibiting and G(1)-phase-blocking activities are reduce d 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-depende nt kinase II (CaMKII), serves to abrogate the inhibitory effect of the extract on DNA synthesis without influencing its ability to induce th e G(1)-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 signa l transduction pathway mediating suppression of DNA synthesis and (2) a p53-dependent pathway mediating G(1)-phase checkpoint control. (C) 1 997 by Radiation Research Society