CHARACTERIZATION OF THE SIGNAL-TRANSDUCTION PATHWAY MEDIATING GAMMA-RAY-INDUCED INHIBITION OF DNA-SYNTHESIS IN HUMAN-CELLS - INDIRECT EVIDENCE FOR INVOLVEMENT OF CALMODULIN BUT NOT PROTEIN-KINASE-C NOR P53

Cultured cells from patients inheriting the rare cancer-prone and radi otherapy-sensitive disorder ataxia-telangiectasia (A-T) exhibit anomal ies in cell. cycle control and protein kinase C (PKC)-mediated upregul ation of p53 protein following exposure to ionizing radiation. It rema ins unclear, however, as to whether this irregularity in a p53-depende nt signal transduction pathway controlling the G(1)/S checkpoint is ca usally linked to the most consistent molecular hallmark. of A-T - name ly, marked attenuation in the inhibition of replicative DNA synthesis at early times (less than or equal to 2 h) after irradiation [radiores istant DNA synthesis (RDS)]. We report here that treatment of normal h uman fibroblast strains with inhibitors of calmodulin (CaM) (i.e. W7 a nd W13) and CaM-dependent protein kinases II and IV (i.e. KN62) prior to radiation exposure elicits an 'A-T-like' RDS phenotype, whereas tre atment with PKC inhibitors (e.g. staurosporine) does not produce this response. Moreover, at 1 h post-gamma irradiation A-T fibroblasts unde rgo normal induction of p53 protein while exhibiting the RDS trait. At later times (e.g. 4 h) following irradiation, however, these A-T cell s contain abnormally low levels of p53 protein, as do their lymphoblas toid cell line counterparts during the entire post-gamma ray incubatio n period. On the other hand, human cells which either lack the p53 gen e completely (i.e. HL60 leukemia cells) or harbor a germline mutation in the gene (i.e. Li-Frauneni syndrome cells) shut down their DNA repl ication machinery normally upon sustaining radiation damage. We thus c onclude that the transitory delay in DNA; synthesis routinely experien ced by human cells in the face of radiation injury is mediated through a CaM-dependent regulatory cascade which involves neither PKC nor p53 protein. Accordingly, A-T cells appear to be malfunctional in at leas t two distinct radiation-responsive signalling pathways, one regulatin g the G(1)/S checkpoint and governed by p53 and PKC and another contro lling passage through S phase and requiring CaM.