PubMed · 7581439
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M Buchwald. 1995. Complementation groups: one or more per gene?. https://doi.org/10.1038/ng1195-228
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It has been reported that the p53 gene mediates an ionizing radiation-induced G1 arrest in mammalian cells. To further characterize this important phenomenon, a panel of seven human diploid fibroblast cell strains and 14 human tumor cell lines from a variety of sources with both wild-type and mutant p53 status were assayed for their susceptibility to G1 arrest after gamma-ray irradiation by a continuous labeling [3H]thymidine incorporation technique. An irreversible G1-block involving 20-70% of the cell population was observed in diploid fibroblasts irradiated with 4 Gy. The block was abolished by transfection with the Human Papilloma Virus E6 gene and in an ataxia telangiectasia (AT) cell line, indicating a role for the AT and p53 genes respectively in this process. In contrast to wild-type normal fibroblast cell strains, the G1-block in all tumor cell lines was significantly reduced, irrespective of their p53 status. None of the nine human tumor cell lines with mutant p53 genes showed a significant G1-block following irradiation with 4 Gy. Among the five tumor cell lines expressing wild-type p53, two showed no apparent G1-block. The remaining three showed a G1-block involving only 8-15% of the cell population, a block much smaller in magnitude than that seen in diploid fibroblasts. Finally, a diploid fibroblast cell strain and a tumor cell line, both showing a normal p53 and p21/WAF1 expression pattern, were examined for pRb phosphorylation before and after irradiation. The diploid fibroblast cell strain showed a significant G1-arrest and a clear inhibition of pRb phosphorylation by irradiation whereas the tumor cells showed no G1-arrest and no inhibition of pRb phosphorylation. These results suggest that (1) multiple genetic factors may modulate the occurrence and magnitude of the G1-arrest induced by exposure to ionizing radiation, (2) the capacity for p53 to mediate a radiation-induced G1 arrest is significantly reduced in tumor cells, (3) the disruption of G1-block modulating factor(s) other than p53 may be an important step in carcinogenesis.
Cultured cells from patients inheriting the rare cancer-prone and radiotherapy-sensitive disorder ataxia-telangiectasia (A-T) exhibit anomalies in cell cycle control and protein kinase C (PKC)-mediated upregulation of p53 protein following exposure to ionizing radiation. It remains unclear, however, as to whether this irregularity in a p53-dependent signal transduction pathway controlling the G1/S checkpoint is causally linked to the most consistent molecular hallmark of A-T-namely, marked attenuation in the inhibition of replicative DNA synthesis at early times (< or = 2 h) after irradiation [radioresistant DNA synthesis (RDS)]. We report here that treatment of normal human fibroblast strains with inhibitors of calmodulin (CaM) (i.e. W7 and W13) and CaM-dependent protein kinases II and IV (i.e. KN62) prior to radiation exposure elicits an 'A-T-like' RDS phenotype, whereas treatment with PKC inhibitors (e.g. staurosporine) does not produce this response. Moreover, at 1 h post-gamma irradiation A-T fibroblasts undergo normal induction of p53 protein while exhibiting the RDS trait. At later times (e.g. 4 h) following irradiation, however, these A-T cells contain abnormally low levels of p53 protein, as do their lymphoblastoid cell line counterparts during the entire post-gamma ray incubation period. On the other hand, human cells which either lack the p53 gene completely (i.e. HL60 leukemia cells) or harbor a germline mutation in the gene (i.e. Li-Fraumeni syndrome cells) shut down their DNA replication machinery normally upon sustaining radiation damage. We thus conclude that the transitory delay in DNA synthesis routinely experienced 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 least two distinct radiation-responsive signalling pathways, one regulating the G1/S checkpoint and governed by p53 and PKC and another controlling passage through S phase and requiring CaM.
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