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Involvement of DNA-dependent protein kinase in down-regulation of cell cycle progression
Fumiaki Watanabe1, Ken-ichi Shinohara, Hirobumi Teraoka
1Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, 101-0062, Tokyo, Japan.
Abstract:
The catalytic polypeptide of DNA-dependent protein kinase (p470) is encoded by the gene responsible for murine severe combined immunodeficiency (SCID) devoid of DNA double-strand break repair and V(D)J recombination. Here, we have characterized the role of p470 in cell proliferation using SCID mice and the cell lines. In accord with DNA histogram patterns, SCID cell lines (SD/SD-eA and SC3VA2) expressing extremely low level of DNA-PK activity grew faster than a normal mouse cell line (CB/CB-eB) and SC3VA2 complemented with human p470 gene (RD13B2). In regenerating liver after partial hepatectomy, de novo DNA synthesis determined by [(3)H]thymidine incorporation started at 30h in C.B-17/Icr-SCID (SCID) mice and at around 36h in C.B-17/Icr (C.B-17) mice. Compared with normal cells, SCID cells contained slightly higher levels of transcripts of cyclin A, cyclin E, B-Myb and dihydrofolate reductase, which are regulated by E2F-1. E2F-1 playing a key role in G1- to S-phase progression was phosphorylated in vitro by DNA-PK. Importantly, the E2F-1 promoter transcriptional activity in SCID cell lines (SD/SD-eA and SC3VA2) was 4-5-fold higher than that in CB/CB-eB and RD13B2. These results suggest that p470 is involved in down-regulation of cell cycle progression through E2F-1-responsible genes.
Insights
DNA-dependent protein kinase (p470) deficiency in SCID mice accelerates cell proliferation by increasing E2F-1 activity. This suggests p470 normally down-regulates cell cycle progression via E2F-1-regulated genes.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Murine severe combined immunodeficiency (SCID) is linked to a deficiency in DNA-dependent protein kinase (DNA-PK) catalytic subunit (p470).
- DNA-PK is crucial for DNA double-strand break repair and V(D)J recombination.
Purpose of the Study:
- To investigate the role of p470 in cell proliferation using SCID mouse models and cell lines.
- To elucidate the mechanism by which p470 influences cell cycle progression.
Main Methods:
- Comparative analysis of cell proliferation rates in SCID and normal mouse cell lines.
- Assessment of de novo DNA synthesis in regenerating livers of SCID and normal mice.
- Quantification of cell cycle regulatory gene transcripts (cyclin A, cyclin E, B-Myb, dihydrofolate reductase) and E2F-1 promoter activity.
Main Results:
- SCID cell lines with low DNA-PK activity exhibited faster proliferation compared to normal cells.
- SCID cells showed higher transcript levels of E2F-1-regulated genes.
- E2F-1 promoter activity was significantly elevated in SCID cells, and DNA-PK phosphorylated E2F-1 in vitro.
- De novo DNA synthesis was initiated earlier in SCID mice liver regeneration.
Conclusions:
- The catalytic polypeptide of DNA-dependent protein kinase (p470) plays a role in down-regulating cell cycle progression.
- p470 likely exerts its effect through the E2F-1 transcription factor and its downstream target genes.
- Deficiency in p470 leads to accelerated cell proliferation due to dysregulation of the G1- to S-phase transition.