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Diminished capacity for p53 in mediating a radiation-induced G1 arrest in established human tumor cell lines
C Y Li1, H Nagasawa, W K Dahlberg
1Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Abstract:
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.
Insights
Ionizing radiation induces a G1 cell cycle arrest in normal cells, mediated by the p53 gene. This radiation-induced G1 arrest is significantly impaired in tumor cells, suggesting other factors contribute to cancer development.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The p53 gene is known to mediate G1 cell cycle arrest in response to ionizing radiation in mammalian cells.
- Understanding the factors regulating this crucial checkpoint is vital for comprehending cellular responses to DNA damage and carcinogenesis.
Purpose of the Study:
- To investigate the role of p53 in mediating G1 arrest following gamma-ray irradiation in human diploid fibroblasts and tumor cell lines.
- To assess the impact of p53 status and other genetic factors on the G1 arrest response to ionizing radiation.
Main Methods:
- Assay of G1 arrest susceptibility using [3H]thymidine incorporation after gamma-ray irradiation.
- Transfection studies with Human Papilloma Virus E6 gene.
- Analysis of p53 and p21/WAF1 expression and pRb phosphorylation.
Main Results:
- Diploid fibroblasts exhibited a significant G1 block after 4 Gy irradiation, which was abolished by HPV E6 or in ataxia telangiectasia (AT) cells.
- Tumor cell lines, regardless of p53 status, showed a markedly reduced G1 block compared to normal fibroblasts.
- Tumor cells with wild-type p53 also displayed diminished or absent G1 arrest and failed to inhibit pRb phosphorylation post-irradiation.
Conclusions:
- Multiple genetic factors influence the G1 arrest response to ionizing radiation.
- The p53-mediated G1 arrest is significantly compromised in human tumor cells.
- Disruption of G1 block regulators, beyond p53, may be a critical step in cancer development.