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Minisatellite instability in severe combined immunodeficiency mouse cells
H Imai1, H Nakagama, K Komatsu
1Carcinogenesis, National Cancer Center Research Institute, 1-1, Tsukiji 5, Chuo-ku, Tokyo 104, Japan.
Abstract:
We have recently found that okadaic acid, which shows strong inhibitory activity on protein serine/threonine phosphatases and tumor-promoting activity in vivo and in vitro, induces minisatellite mutation (MSM). Human tumors and chemically induced counterparts in experimental animals are also sometimes associated with MSM. In the present study, we demonstrated minisatellite (MS) instability in severe combined immunodeficiency (SCID) cells in which the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is impaired. Cells from a SCID fibroblast cell line transformed by simian virus 40 large tumor antigen, SC3VA2, and from an embryonal SCID fibroblast cell line, SC1K, were cloned and propagated to 10(7) to 10(8) cells, and then subjected to subcloning. After propagation of each subclone to 10(7) to 10(8) cells, DNA samples were digested with HinfI and analyzed by Southern blotting using the Pc-1 MS sequence as a probe. Under low-stringency conditions, about 40 MS bands were detected, with 45% +/- 6% and 37% +/- 3% of SC3VA2 and SC1K cells, respectively, having MSM. In contrast, cells from the RD13B2 cell line, which was established from SCVA2 by introducing human chromosome 8q fragments, on which DNA-PKcs is known to reside, to complement the SCID phenotype, showed a very low frequency of MSM (3% +/- 3%). The high frequencies of MSM in SC3VA2 and SC1K were significant, with no difference between the two. The present study clearly demonstrates that MS instability exists in SCID fibroblasts, suggesting that DNA-PKcs might be involved in the stable maintenance of MS sequences in the genome.
Insights
Minisatellite mutation (MSM) is linked to impaired DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in severe combined immunodeficiency (SCID) cells. This suggests DNA-PKcs plays a crucial role in maintaining minisatellite sequence stability in the genome.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Minisatellite mutations (MSM) are observed in human tumors and experimental models.
- Okadaic acid induces MSM and exhibits tumor-promoting activity.
- Severe combined immunodeficiency (SCID) is characterized by impaired DNA repair pathways.
Purpose of the Study:
- To investigate the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in minisatellite (MS) stability.
- To determine if MS instability is present in SCID cells with impaired DNA-PKcs.
Main Methods:
- Utilized SCID fibroblast cell lines (SC3VA2, SC1K) with impaired DNA-PKcs.
- Subcloned cells and analyzed DNA for minisatellite instability using Southern blotting.
- Compared MSM frequencies in SCID cells with complemented cell lines (RD13B2).
Main Results:
- SCID cells (SC3VA2, SC1K) exhibited high frequencies of minisatellite mutation (MSM) (45% and 37%, respectively).
- Complementation of DNA-PKcs in RD13B2 cells significantly reduced MSM frequency to 3%.
- No significant difference in MSM was observed between the two SCID cell lines.
Conclusions:
- Minisatellite instability is evident in SCID fibroblasts lacking functional DNA-PKcs.
- DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is implicated in the stable maintenance of minisatellite sequences.
- Findings suggest a potential link between DNA repair mechanisms and genome stability at minisatellite loci.