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Altered DNA repair and dysregulation of p53 in IRF-1 null hepatocytes
S Prost1, C O Bellamy, D S Cunningham
1Department of Pathology, University Medical School, Edinburgh, Scotland. s.prost@ed.ac.uk
Abstract:
The tumor suppressor proteins IRF-1 and p53 are involved in response pathways after DNA damage. In different cell types, IRF-1 and p53 can cooperate to produce cell cycle arrest (embryo fibroblasts) or can independently trigger apoptosis (lymphoid cells). p53 may also regulate DNA repair, but there is no information on IRF-1 and repair. The cell lineage dependency of these effects precludes extrapolation of findings to other tissues of relevance to human cancer. Here, we report the consequences of IRF-1 deficiency for apoptosis, cell cycle arrest, and DNA repair in primary hepatocytes after DNA damage and extend previous work on the role of p53 in hepatocytes. IRF-1-deficient hepatocytes showed reduced DNA repair activity compared with wild-type, as assessed by unscheduled DNA synthesis after UV irradiation (10J/m2) and by host reactivation of a UV-damaged reporter construct. p53-deficient hepatocytes also showed reduced unscheduled DNA synthesis after UV, but there was no impairment of specific repair in host reactivation assays. IRF-1 deficiency did not affect the p53-dependent G1/S arrest after UV irradiation. Hepatocyte apoptosis after UV treatment, previously reported to be independent of p53, was also independent of IRF-1. However, IRF-1 deficiency produced dysregulation of p53, manifested as increased transactivation of a p53-reporter plasmid in undamaged hepatocytes, and accelerated p53 stabilization after DNA damage. Hence, in hepatocytes, IRF-1 is not required for growth arrest or apoptosis after DNA damage, but the results suggest for the first time a role in DNA repair regulation.
Insights
Interferon regulatory factor-1 (IRF-1) deficiency impairs DNA repair in hepatocytes but does not affect apoptosis or cell cycle arrest. IRF-1 dysregulates p53 stability and activity following DNA damage.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Tumor suppressor proteins Interferon regulatory factor-1 (IRF-1) and p53 are crucial in DNA damage response pathways.
- Their roles in cell cycle arrest and apoptosis vary significantly across different cell types.
- The involvement of IRF-1 in DNA repair remains largely uncharacterized, particularly in liver cells.
Purpose of the Study:
- To investigate the consequences of IRF-1 deficiency on apoptosis, cell cycle arrest, and DNA repair in primary hepatocytes following DNA damage.
- To extend the understanding of p53's role in hepatocyte DNA damage response.
- To elucidate the novel role of IRF-1 in DNA repair regulation within hepatocytes.
Main Methods:
- Primary hepatocytes were subjected to UV irradiation (10J/m2) to induce DNA damage.
- DNA repair activity was assessed using unscheduled DNA synthesis (UDS) assays.
- Host cell reactivation assays were employed to evaluate the repair of a UV-damaged reporter construct.
- p53-dependent G1/S arrest and apoptosis were analyzed in IRF-1 and p53 deficient hepatocytes.
- p53 transactivation and stabilization were measured using reporter plasmids and Western blotting.
Main Results:
- IRF-1-deficient hepatocytes exhibited significantly reduced DNA repair capacity compared to wild-type cells, as evidenced by lower UDS and impaired host reactivation.
- p53-deficient hepatocytes also showed diminished UDS, but specific repair mechanisms were not affected in host reactivation assays.
- IRF-1 deficiency did not alter the p53-dependent G1/S cell cycle arrest or apoptosis induction following UV irradiation.
- IRF-1 deficiency led to dysregulation of p53, characterized by increased transactivation in undamaged cells and accelerated stabilization after DNA damage.
Conclusions:
- In hepatocytes, IRF-1 is not essential for mediating cell cycle arrest or apoptosis subsequent to DNA damage.
- The study provides the first evidence suggesting a role for IRF-1 in the regulation of DNA repair processes in hepatocytes.
- IRF-1 deficiency influences p53 activity and stability, highlighting an intricate interplay between these tumor suppressors in DNA damage response.
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